Docosahexaenoic acid-containing oil and method for producing same

ABSTRACT

Docosahexaenoic acid-containing oil containing docosahexaenoic acid in a concentration of 40 wt. % or more of the total weight of fatty acids in the oil, and having an endothermic peak temperature determined by differential scanning calorimetry (DSC) of 15° C. or lower; a biomass including the same; and a method for producing docosahexaenoic acid-containing oil including obtaining a biomass by culturing microorganisms of the genus  Aurantiochytrium  capable of producing this docosahexaenoic acid-containing oil, recovering the biomass after culture, and extracting the oil from the biomass after recovery.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation application of International Patent ApplicationNo. PCT/JP2016/085637 filed Nov. 30, 2016, which claims the benefit ofJapanese Patent Application No. 2015-234985, the full contents of all ofwhich are hereby incorporated by reference in their entirety.

BACKGROUND Technical Field

The present disclosure relates to a docosahexaenoic acid containing oiland a manufacturing method thereof.

Description of the Related Art

Eicosapentaenoic acid (hereinafter, also abbreviated as EPA),docosahexaenoic acid (hereinafter, also abbreviated as DHA) and the likeare n−3 unsaturated fatty acids (hereinafter, n−3 PUFA) that havevarious physiological effects and are utilized as medicaments, healthfood products, food materials, and the like. Beverages obtained byadding fish oils including DHA are recognized as food for specifiedhealth uses. There is a very high demand for n−3 PUFA as a supplementinside and outside Japan. Accordingly, there has been a demand for theproduction of polyunsaturated fatty acids in large quantities at highpurities. In order to industrially efficiently obtain DHA as one suchn−3 PUFA, while techniques for concentrating DHA in DHA containing oilinto a high content using distillation techniques, and the like areused, it has been proposed to obtain an oil containing high content DHAas a raw material oil prior to applying such concentration techniques.

For example, JP 2015-509733 A discloses microorganisms which yieldlipids containing higher than 40% DHA in a fatty acid composition byculturing microbial strains belonging to the genus Schizochytrium underculture conditions in combination with predetermined lightingconditions.

JP H10-72590 A discloses that a DHA containing oil having a DHA contentof higher than 40% is obtained from a specific microorganisms belongingto of the Thraustochytrium isolated as marine microorganisms.

In oil and fat for food products, the endothermic peak determined bydifferential scanning calorimetry (DSC) is measured as an index withrespect to the behavior of crystallization (for example, JP H06-14710 Aand JP 2005-507028 A).

SUMMARY

Even an oil containing high content DHA may exhibit differentproperties, due to the presence of saturated or unsaturated fatty acidsother than DHA. For example, even in a case where an oil contains DHA atthe same high content and the temperature decreases from roomtemperature, in accordance with the kind and concentration of othersaturated or unsaturated fatty acids, the properties of the DHAcontaining oil at lower temperatures change or crystallization occurs,causing the oil to solidify and consequently resulting in a reduction inhandleability exemplified by pipe clogging.

Even a biomass containing high content DHA may exhibit differentproperties, due to the presence of saturated or unsaturated fatty acidsother than DHA. For example, even regarding a biomass containing an oilcontaining DHA at the same high content, the properties of the entirebiomass change in accordance with the kind and concentration of othersaturated or unsaturated fatty acids, the viscosity increases, andconsequently, the handleability may be reduced such that clogging of anapparatus such as an extruder tends to occur during kneading under highpressure, and the like.

The object of the present disclosure is to provide a docosahexaenoicacid containing oil and biomass which contain a high contentdocosahexaenoic acid and have excellent stability at low temperatures,along with the manufacturing method thereof.

The present disclosure includes each of the following aspects.

[1] A docosahexaenoic acid containing oil, the oil includingdocosahexaenoic acid at a concentration of 40 wt. % or more of the totalweight of fatty acid in the oil, with the endothermic peak temperaturethereof determined by differential scanning calorimetry (DSC) being 15°C. or lower.

[2] The docosahexaenoic acid containing oil according to [1], whereinthe endothermic peak temperature determined by differential scanningcalorimetry (DSC) is 10° C. or lower.

[3] The docosahexaenoic acid containing oil according to [1] or [2],wherein the docosahexaenoic acid concentration is from 40 wt. % to 98wt. % of the total weight of fatty acid in the oil.

[4] The docosahexaenoic acid containing oil according to any one of [1]to [3], wherein the endothermic peak temperature determined bydifferential scanning calorimetry (DSC) is from −50° C. to 10° C.

[5] The docosahexaenoic acid containing oil according to any one of [1]to [4], wherein the oil is a microbial oil.

[6] The docosahexaenoic acid containing oil according to [5], whereinthe microbial oil is a microbial oil of at least one microorganismselected from the group consisting of Opisthokonta, Archaeplastida,Excavata, SAR, microorganisms belonging to Haptophyta and Cryptophytanot classified as the aforementioned, and bacterium.

[7] The docosahexaenoic acid containing oil according to [5], whereinthe microbial oil is a microbial oil from microorganisms belonging toStramenopiles.

[8] The docosahexaenoic acid containing oil according to [5], whereinthe microbial oil is a microbial oil from microorganisms belonging tothe class Labyrinthulea.

[9] The docosahexaenoic acid containing oil according to [5], whereinthe microbial oil is a microbial oil from microorganisms belonging toThraustochytrid microorganisms.

[10] The docosahexaenoic acid containing oil according to [5], whereinthe microbial oil is a microbial oil from microorganisms of the genusAurantiochytrium.

[11] The docosahexaenoic acid containing oil according to [5], whereinthe microbial oil is a microbial oil of a docosahexaenoic acid producingmutant of Aurantiochytrium limacinum.

[12] The docosahexaenoic acid containing oil according to [5], whereinthe microbial oil is microbial oil of the Aurantiochytrium limacinumNiD2 strain (accession number FERM BP-22296), the NiD3 strain (accessionnumber FERM BP-22297), or a microbial strain having substantially thesame microbiological properties as the strain.

[13] The docosahexaenoic acid containing oil according to any one of [1]to [12], wherein the oil is a crude oil of the microbial oil.

[14] The docosahexaenoic acid containing oil according to any one of [1]to [12], wherein the oil is a refined oil of the microbial oil.

[15] A biomass of microorganisms, the biomass including thedocosahexaenoic acid containing oil according to any one of [1] to [13].

[16] The biomass according to [15], wherein the accumulative amount ofthe docosahexaenoic acid is 18. wt % or more of the dried biomass weightper 1 liter of a culture product.

[17] The biomass according to [15] or [16], wherein the biomass is adried biomass.

[18] Microorganisms of the genus Aurantiochytrium capable of producingthe docosahexaenoic acid containing oil according to any one of [1] to[14].

[19] The microorganisms of the genus Aurantiochytrium according to [18],wherein the microorganisms are of the Aurantiochytrium limacinum NiD2strain (accession number FERM BP-22296) or the NiD3 strain (accessionnumber FERM BP-22297).

[20] The microorganisms of the genus Aurantiochytrium according to [19],having substantially the same microbiological properties as the NiD2strain (accession number FERM BP-22296) or the NiD3 strain (accessionnumber FERM BP-22297).

[21] A manufacturing method of the biomass according to any one of [15]to [17], the method including: culturing the microorganisms of the genusAurantiochytrium according to any one of [18] to [20] to obtain abiomass; and recovering the cultured biomass.

[22] The manufacturing method of biomass according to [21], the methodfurther including drying the biomass obtained after culture.

[23] A manufacturing method of docosahexaenoic acid containing oil, themethod including: culturing the microorganisms of the genusAurantiochytrium according to any one of [18] to [20] to obtain abiomass; recovering the cultured biomass; and extracting oil from therecovered biomass, wherein a docosahexaenoic acid containing oilcomprising docosahexaenoic acid at a concentration of 40 wt. % or moreof the total weight of fatty acid in the oil is obtained from theextracted oil with the endothermic peak temperature thereof determinedby differential scanning calorimetry (DSC) being 15° C. or lower.

[24] The manufacturing method according to [23], wherein the obtaineddocosahexaenoic acid containing oil is a crude oil.

[25] The manufacturing method according to [23], the method furtherincluding refining the extracted oil.

[26] The manufacturing method according to any one of [21] to [25],wherein the microorganisms are cultured at 10° C. to 40° C.

[27] A food product, supplement, medicament, cosmetic, or animal feedcontaining the docosahexaenoic acid containing oil according to any oneof [1] to [14] or the biomass according to any one of [15] to [17].

[28] Use of the docosahexaenoic acid containing oil according to any oneof [1] to [14] or the biomass according to any one of [15] to [17] in amanufacturing method of a food product, supplement, medicament,cosmetic, or animal feed.

According to the present disclosure, a docosahexaenoic acid containingoil and a biomass which contain a high content docosahexaenoic acid andhave excellent stability at low temperatures, along with themanufacturing method thereof.

DETAILED DESCRIPTION

The docosahexaenoic acid containing oil in one embodiment is an oilincluding docosahexaenoic acid at a concentration of 40 wt. % or more ofthe total weight of fatty acid in the oil with the endothermic peaktemperature thereof determined by differential scanning calorimetry(DSC) being 15° C. or lower. The biomass of microorganisms in oneembodiment is a biomass including this docosahexaenoic acid containingoil, and microorganisms in one embodiment are microorganisms which mayproduce this docosahexaenoic acid containing oil.

Even in a case where the docosahexaenoic acid containing oil in thisembodiment contains a high content docosahexaenoic acid, in addition toincluding components other than the docosahexaenoic acid in the oil, forexample, other saturated or unsaturated fatty acids, the properties tendnot to change, with the speed and behavior of crystallization moderatelyadjusted and a tendency seen to not solidify at low temperatures.Therefore, the docosahexaenoic acid containing oil in this embodimentcan have excellent stability at low temperatures, have a suitableviscosity, and exhibit smooth properties over a wide temperature range.Consequently, the docosahexaenoic acid containing oil in itself canexhibit favorable handleability, and, can eliminate the need to combineadditives such as a viscosity adjustor, additionally. The biomass anddried biomass according to this embodiment retain an oil containing thedocosahexaenoic acid according to this embodiment in the biomass,thereby making them favorable to efficiently obtain the docosahexaenoicacid containing oil containing high content DHA and having excellentstability at low temperatures. Low temperatures in the presentspecification mean room temperature, that is, a temperature of 25° C. orlower, and for example, refers to 15° C. or lower, 10° C. or lower, or5° C. or lower, and possibly 0° C. or lower.

In the present specification, the docosahexaenoic acid containing oilaccording to one embodiment containing DHA at the specific concentrationand having the specific endothermic peak temperature thereof determinedby DSC is also simply referred to as a “high content DHA containingoil.”

In the present specification, in addition to an independent step, theterm “step” also refers to a step that achieves the intended object ofthe step even when the step cannot be clearly distinguished from othersteps. In the present specification, numeric ranges indicated by “to”are ranges that include the minimum and maximum values each statedbefore and after “to.” In the present specification, the terms “orlower” or “or less” and “lower than” or “less than” in regard topercentages mean ranges including 0% or a value undetectable by thepresent means, unless the lower limit is specifically stated.

In the present specification, for the case in which multiple substancescorresponding to each of the components in the mixture are present, theamount of each component in the composition, unless otherwise noted, istaken to mean the total amount of these multiple substances present inthe mixture. In the present specification, for the case in whichmultiple substances corresponding to each of the components in themixture are present, the concentration or content (%) of each componentin the composition, unless otherwise noted, is taken to mean the totalconcentration or content of these multiple substances present in themixture.

In the present specification, unless otherwise noted, when a numericalrange that only specifies one or more upper limit values and a numericalrange that only specifies one or more lower limit values are describedfor an identical target, the embodiment of the present disclosureincludes a numerical range having a combination of any upper limit valuechosen from the one or more upper limit values and any lower limit valuechosen from the one or more lower limit values.

In the present specification, the terms “oil” and “oil and fat” refer toa mixture of organic substances which is insoluble in water at roomtemperature and normal pressure, that is, at 25° C. at 1 atm, includesoils containing only triglycerides and also includes crude oilscontaining triglycerides as a main component and other lipid such asdiglycerides, monoglycerides, phospholipids, cholesterol, and free fattyacids. “Oil” and “oil and fat” mean compositions containing theselipids. In the present specification, the term “crude oil” means amixture of the abovementioned lipids and means an oil in the stateobtained by extraction from an organism. In the present specification,the term “refined oil” means an oil obtained via a refining process inwhich a crude oil is subjected to at least one oil and fat refining stepselected from the group consisting of a degumming step, deacidificationstep, decoloring step, and deodorizing step to remove substances such asphospholipids and sterol other than the target.

In the present specification, a “microbial oil” means an oil obtainedusing microbial biomass as a source. Exemplary microbial oils includeoily components such as saturated or unsaturated fatty acids,phospholipids, sterols, glycerol, ceramides, sphingolipids, terpenoids,flavonoids, and tocopherols. The saturated or unsaturated fatty acid maybe present as a constituent fatty acid in other oily components.

In the present specification, the term “fatty acid” not only indicates afree saturated or unsaturated fatty acid itself, but also includes fattyacids contained as constituent units in free saturated or unsaturatedfatty acids, saturated or unsaturated fatty acid alkyl esters,triglycerides, diglycerides, monoglycerides, phospholipids, sterylesters, and the like, which can also be called constituent fatty acids.

In the present specification, unless otherwise noted or indicated, whena fatty acid that is present or used is mentioned, the presence or useof fatty acid containing compounds in any form is included. Exemplaryforms of compounds containing fatty acids may include a free fatty acidform, fatty acid alkyl ester form, glyceryl ester form, phospholipidform, and steryl ester form. When a fatty acid is specified, one formmay be present or a mixture of two or more forms may be present.

When denoting fatty acids, a numerical expression may be used, whereinthe number of carbon, the number of double bonds, and the locations ofdouble bonds are each expressed in a simplified manner using numbers andalphabets. For example, a saturated fatty acid having 20 carbons may benotated as “C20:0.” A monounsaturated fatty acid having 18 carbons maybe notated as “C18:1” and the like. Dihomo-γ-linolenic acid may benotated as “C20:3, n−6” and the like. Arachidonic acid may be expressedas “C20:4, n−6” and the like. Note that “n−6” is also denoted as ω-6,with this indicating that the bonding position of a first double bond isat the sixth position when the position is counted from the last carbon(ω) to the carboxy group. This method is known to those skilled in theart and those skilled in the art can easily specify fatty acidsexpressed in accordance with this method.

The fatty acid concentration in the present specification is determinedbased on the fatty acid composition unless otherwise noted. Thecomposition of fatty acids may be determined by standard methods.Specifically, a fatty acid lower alkyl ester obtained by esterifying theoil to be measured using a lower alcohol and a catalyst is used. Next,the obtained fatty acid lower alkyl ester is analyzed as a sample usinga gas chromatograph with a flame ionization detector (FID). Peakscorresponding to each of the fatty acids are identified in the obtainedgas chromatography chart, with the peak area of each of the fatty acidsdetermined using the Agilent ChemStation integration algorithm (revisionC.01.03[37], Agilent Technologies). The percentage (area %) of each peakarea to the total sum of the peak area of the fatty acids is the fattyacid composition. The value according to the area % obtained by theabovementioned measurement method can be used interchangeably so as tobe the same as the value according to the wt. % of each fatty acid in asample, and regarding the fatty acid composition in the presentdisclosure, the area % obtained by the abovementioned measurement methodis defined as the notation of “wt. %”. Refer to “Basic Oil AnalyticalTest Methods”, 2013 Edition, 2.4.2.1-2013 Fatty Acid Composition (FIDconstant temperature gas chromatograph method) and 2.4.2.2-2013 FattyAcid Composition (FID heating gas chromatograph method) established bythe Japan Oil Chemists' Society (JOCS).

In the present specification, the term “biomass” means an aggregation ormass of cells at a certain point of time during growth in a certainregion or in an ecosystem. This region or ecosystem may be a naturallypresent environment, for example, a water area, or may be a synthesisenvironment, for example, an open type or sealed type fermentation tankor bioreactor. In the present specification, a biomass obtained after adrying step is also particularly referred to as a “dried biomass.”

(1) High Content DHA Containing Oil

The high content DHA containing oil includes DHA at a concentration of40 wt. % or more of the total weight of fatty acid in the oil and theendothermic peak temperature thereof determined by differential scanningcalorimetry (DSC) is 15° C. or lower.

The DHA concentration in the high content DHA containing oil can be 40wt. % or more, 43 wt. % or more, 50 wt. % or more, 55 wt. % or more, 60wt. % or more, 65 wt. % or more, or 70 wt. % or more of the total weightof fatty acid in the oil. The DHA concentration in the high content DHAcontaining oil may be 98 wt % or less, 90 wt. % or less, or 80 wt. % orless of the total weight of fatty acid in the oil. The upper limit valueand lower limit value of the DHA concentration in the high content DHAcontaining oil may be any of the following combinations. For example,the DHA concentration of the high content DHA containing oil may be from40 wt. % to 98 wt. %, from 43 wt. % to 98 wt. %, from 50 wt. % to 98 wt.%, from 60 wt. % to 98 wt. %, from 65 wt. % to 98 wt. %, or from 70 wt.% to 98 wt. % of the total weight of fatty acid in the oil. As the DHAconcentration in the high content DHA containing oil increases, thefunction of DHA in the high content DHA containing oil can be expectedto be more strongly exerted.

The high content DHA containing oil can contain a fatty acid other thanDHA. Hereinafter, fatty acids other than DHA contained in the highcontent DHA containing oil are also referred to as “low content fattyacids.” The low content fatty acid may be saturated fatty acid orunsaturated fatty acid, with one or a combination of multiple kindscapable of being contained in the oil. The low content fatty acid can beless than 60 wt. %, more than 2 wt. % and less than 60 wt. %, from 5 wt.% to 50 wt. %, or from 5 wt. % to 30 wt. % of the total weight of fattyacid in the oil. The low content fatty acid concentration can be presentat a higher concentration than DHA in the total concentration in a casewhere multiple kinds are combined, and in this case, each concentrationof low content fatty acids is preferably no higher than the DHAconcentration. In terms of the stability of the high content DHAcontaining oil at low temperatures, regarding the high content DHAcontaining oil, the total concentration of the low content fatty acidpresent in the oil is preferably no higher than the DHA concentration.In a case where the low content fatty acid is within this range, theviscosity of the DHA containing oil at low temperatures is suitable andthe handleability tends not to significantly deteriorate.

Exemplary unsaturated fatty acids which may be present as the lowcontent fatty acid may include di- or higher, preferably tri- or higherunsaturated fatty acid having 20 or more carbons. Exemplary tri- orhigher unsaturated fatty acids having 20 or more carbons may includepolyunsaturated fatty acids having from 20 to 22 carbons. Specificexamples thereof include eicosadienoic acid (C20:2, n-9, EDA),dihomo-γ-linolenic acid (C20:3, n−6, DGLA), Mead acid (C20:3, n-9, MA),eicosatetraenoic acid (C20:4, n−3, ETA), arachidonic acid (C20:4, n−6,ARA), eicosapentaenoic acid (C20:5, n−3, EPA), docosatetraenoic acid(C22:4, n−6, DTA), docosapentaenoic acid (C22:5, n−3, _(n-3)DPA),docosapentaenoic acid (C22:5, n-6, _(n-6)DPA), and the like.

For example, the high content DHA containing oil can include _(n-6)DPA,for example, 5 wt. % or more or 8 wt. % or more of the total weight offatty acid in the oil, and can be 20 wt. % or less, or 12 wt. % or less.In a case where the content of _(n-6)DPA in the high content DHAcontaining oil is within this range, the viscosity of the DHA containingoil at low temperatures tends to be suitable and the handleability tendsnot to significantly deteriorate.

The total concentration of the unsaturated fatty acids which may bepresent as these low content fatty acids can include 5 wt. % or more or8 wt. % or more of the total weight of fatty acid in the oil, and can be30 wt. % or less, 20 wt. % or less, or 12 wt. % or less. The highcontent DHA containing oil, in which the total concentration of the lowcontent fatty acids which may be present as unsaturated fatty acids is30 wt. % or less, can be preferably implemented for use such that DHA isdesirably efficiently obtained and stability at low temperatures may befurther improved.

Exemplary saturated fatty acid, which may be present as the low contentfatty acid, may include saturated fatty acids having 12 or more carbons.Exemplary saturated fatty acids having 12 or more carbons may includelauric acid (C12:0), myristic acid (C14:0), palmitic acid (C16:0),stearic acid (C18:0), arachidic acid (C20:0), behenic acid (C22:0),lignoceric acid (C24:0), and the like. In a case where the concentrationof these saturated fatty acids is limited to a predetermined range, thehigh content DHA containing oil has more favorable stability at lowtemperatures, with superior handleability.

The lauric acid (C12:0) concentration in the high content DHA containingoil can be 1.0 wt. % or less, 0.5 wt. % or less, 0.3 wt. % or less, or0.1 wt. %. The concentration ratio of the lauric acid and DHA in thehigh content DHA containing oil, as a ratio of lauric acid/DHA, can be0.3 or less, 0.25 or less, or 0.2 or less. The lower limit value of thecontent of the lauric acid in the high content DHA containing oil maybe, for example, 0.05 wt. %.

The myristic acid (C14:0) concentration in the high content DHAcontaining oil can be 10.0 wt. % or less, 8.0 wt. % or less, 5.0 wt. %or less, 3.0 wt. % or less, or 2.5 wt. % or less. The concentrationratio of the myristic acid and DHA in the high content DHA containingoil, as a ratio of myristic acid/DHA, can be 0.3 or less, 0.2 or less,or 0.1 or less. The lower limit value of the content of the myristicacid in the high content DHA containing oil may be, for example, 0.1 wt.%.

The palmitic acid (C16:0) concentration in the high content DHAcontaining oil can be 40.0 wt. % or less, 35.0 wt. % or less, or 30.0wt. % or less. The concentration ratio of the palmitic acid and DHA inthe high content DHA containing oil, as a ratio of palmitic acid/DHA,can be 0.8 or less, or 0.6 or less. The lower limit value of the contentof the palmitic acid in the high content DHA containing oil may be, forexample, 5.0 wt. %.

The stearic acid (C18:0) concentration in the high content DHAcontaining oil can be 18.0 wt. % or less, 15.0 wt. % or less, 10.0 wt. %or less, 5.0 wt. % or less, 2.0 wt. % or less, or 1.5 wt. %. Theconcentration ratio of the stearic acid and DHA in the high content DHAcontaining oil, as a ratio of stearic acid/DHA, can be 0.5 or less, 0.3or less, or 0.1 or less. The lower limit value of the content of thestearic acid in the high content DHA containing oil may be, for example,0.3 wt. %.

In the high content DHA containing oil, the endothermic peak temperaturethereof determined by DSC is 15° C. or lower. In a case where theendothermic peak temperature determined by DSC is 15° C. or lower, thehigh content DHA containing oil can exhibit suitable viscosity andfavorable handleability. For example, in terms of the viscosity forblending the high content DHA containing oil in other compositions, itis advantageously easily blended. The endothermic peak temperaturedetermined by DSC of the high content DHA containing oil is alsoreferred to as the “DSC pour point” in the present specification.

The DSC pour point is measured as follows.

A measurement sample of 10 mg is weighed in a sample pan using adifferential scanning calorimetry apparatus DSC 3500 Sirius (availablefrom NETZSCH), heated at 50° C. for 5 minutes, cooled at a cooling rateof 3° C./minute from 50° C. to −60° C., and subsequently, thetemperature is raised at a temperature elevation rate of 10° C./minutefrom −60° C. to 50° C. for measurement. The temperature at the maximumvalue of the endothermic peak in the obtained DSC curve is the DSC pourpoint (° C.).

The DSC pour point of the high content DHA containing oil may be 10° C.or lower, 5° C. or lower, 0° C. or lower, −5° C. or lower, or −10° C. orlower. While not particularly limited thereto, the lower limit value ofthe DSC pour point of the high content DHA containing oil can be, forexample, −65° C. or −50° C. In a case where the upper limit value of theDSC pour point is lower, the stability at low temperatures tends to besuperior and to be improved handleability. While not limited to anyspecific theory, in a case where the upper limit value of the DSC pourpoint is lower, the concentration of saturated fatty acids having from12 to 20 carbons tends to be low, and consequently, handleability at lowtemperatures is presumably improved. The upper limit value and lowerlimit value of the DSC pour point of the high content DHA containing oilmay be any of the following combinations. For example, they can be from−65° C. to 15° C., from −50° C. to 10° C., from −50° C. to 5° C., from−50 to 0° C., or from −50° C. to −5° C.

Regarding the high content DHA containing oil, the content of insolublecomponents in the oil can be 10 wt. % or less, 5 wt. % or less, or 3 wt.% or less. In a case where the insoluble components in the oil are 10wt. % or less, the high content DHA containing oil has high clearness aswell as excellent handleability. Insoluble components in high contentDHA containing oil means precipitates which are produced when the highcontent DHA containing oil is left to stand at 25° C. for 1 hour. Theamount of insoluble components in the high content DHA containing oil ismeasured as follows.

One thousand milligram of the high content DHA containing oil iscollected in a weighed micro test tube having a capacity of 2 mlavailable from Eppendorf, left to stand at 25° C. for 1 hour, afterwhich the precipitate is recovered using a trace high speed coolingcentrifuge and the precipitate weight is measured using a precisionbalance to be calculated as wt. % in 1000 mg of the microbial oil. Asthe trace high speed cooling centrifuge, MX-300 available from TomySeiko Co., Ltd. is used.

Derivation of the high content DHA containing oil is not particularlylimited as long as it has the abovementioned predetermined DSC pourpoint and DHA concentration. The high content DHA containing oil can bea bio-oil derived from organisms such as plants, fish, andmicroorganisms, and can be, for example, a microbial oil. A bio-oilmeans an oil obtained using a biomass originating from organisms, whilea microbial oil, as described above, means an oil obtained using abiomass originating from microorganisms. The organisms providing bio-oilmay be genetically modified materials.

The high content DHA containing oil can be a crude oil or a refinedmicrobial oil. In the present specification, “a crude oil of a microbialoil” refers to a composition which is extracted from a biomass ofmicroorganisms and does not undergo the below mentioned refining step.In the present specification, “a refined oil of microbial oil” refers toa composition obtained via a crude oil refining process in which a crudeoil from microorganisms is subjected to a degumming step,deacidification step, decoloring step using an activated clay or activecarbon, washing step, deodorizing step by steam distillation, and thelike to remove substances such as phospholipids and sterols other thanthe target.

The microorganisms may be microorganisms which may produce high contentDHA containing oil, with exemplary microorganisms potentially includingOpisthokonta, Archaeplastida, Excavata, SAR, microorganisms belonging toHaptophyta and Cryptophyta not classified as these, and bacterium, inthe classification system (Adl et. al., The Journal of EukaryoticMicrobiology. 59(5):429-493(2012)) proposed by Adl et. al. Thesemicroorganisms may be used alone or in combination of two or morethereof. That is, among these microorganisms, the microbial oil may be amicrobial oil obtained from one microorganism or may be a mixture ofmicrobial oils obtained from two or more microorganisms.

Exemplary Opisthokonta may include microorganisms belonging to fungi.Exemplary fungi may include Yeast and filamentous fungi. Exemplary Yeastmay include at least one microorganism selected from the groupconsisting of microorganisms of the genus Yarrowia, microorganisms ofthe genus Pichia, microorganisms of the genus Saccharomyces,microorganisms of the genus Cryptococcus, and microorganisms of thegenus Trichospora. Exemplary filamentous fungi may include at least onemicroorganism selected from the group consisting of microorganisms ofthe genus Mortierella, microorganisms of the genus Pythium, andmicroorganisms of the genus Phytophthora. Of these, microorganismsbelonging to the genus Mortierella are even more preferable. Exemplarymicroorganisms belonging to the genus Mortierella include Mortierellaelongata, Mortierella exigua, Mortierella hygrophila, and Mortierellaalpina.

Exemplary Archaeplastida may include microorganisms of Chlorophycea.Exemplary microorganisms of Chlorophycea may include microorganismsbelonging to the genera Botryococcus, Pseudochoricystis, Scenedesmus,and Desmodesmus.

Exemplary Excavata may include microorganisms of Euglenida. Exemplarymicroorganisms of the class Euglenida may include microorganismsbelonging to the family Euglenaceae. Exemplary microorganisms belongingto the family Euglenaceae may include microorganisms belonging to thegenus Euglena.

SAR corresponds to microorganisms of Stramenopiles, Alveolata, andRhizaria. Exemplary Stramenopiles may include at least one microorganismselected from the group consisting of Bicosoecida, Labyrinthulea,Blastocystis, Actinophyida, Opalinata, Placidida, Oomycetes,Hyphochytriomycetes, Developayella, Chrysophyceae, Eustigmatophyceae,Phaeothamniophyceae, Pinguiophyceae, Raphidophyceae, Synurophyceae,Xantexyophyceae, Phaeophyceae, Schizocladiophyceae, Chrysomerophyceae,Dictyochophyceae, Bolidophiceae, Pelagophyceae, and Bacillariophyceaemicroorganisms. Exemplary Alveolata may include microorganisms belongingto Ciliophora, Apicomplexa, and Dinoflagellates. ExemplaryDinoflagellates may include microorganisms belonging to the genusCrypthecodinium. Exemplary microorganisms belonging to the genusCrypthecodinium may include Crypthecodinium cohnii.

Of these, microorganisms belonging to the class Labyrinthulea arepreferable as microorganisms, with microorganisms belonging toThraustochytrid particularly preferable. As microorganisms belonging toThraustochytrid, for example, at least one microorganism selected fromthe group consisting of microorganisms belonging to the generaAplanochytrium, Japonochytrium, Labyrinthuloides, Schizocytrium,Thraustochytrium, Ulkenia, Aurantiochytrium, Oblongichytrium,Botryochytrium, Parietichytrium, and Sicyoidochytrium can be preferablyselected, among which microorganisms of the genus Aurantiochytrium areparticularly preferably selected.

As microorganisms of the genus Aurantiochytrium, at least one selectedfrom the group consisting of Aurantiochytrium limacinum andAurantiochytrium mangrovei can be selected, with Aurantiochytriumlimacinum particularly selected in terms of growth of microorganisms.

The microorganisms belonging to Haptophyta and Cryptophyta correspond tomicroorganisms belonging to Haptophyta and Cryptophyta which do notbelong to any of Opisthokonta, Archaeplastida, Excavata, or SAR.Specific examples thereof may include the classes Haptophyceae andCryptophyceae.

Exemplary bacteria may include microorganisms of Proteobacteria andFirmicutes. Exemplary microorganisms belonging to Proteobacteria mayinclude Escherichia coli. Exemplary microorganisms belonging toFirmicutes may include Bacillus subtilis.

In a case where Stramenopiles are selected as microorganisms for DHAproduction among the abovementioned microorganisms, DHA productionefficiency tends to be further improved. Moreover, marine microorganismspresent in the ocean or brackish water regions among Stramenopiles haverelatively high DHA production efficiency. Among marine microorganisms,microorganisms of the genus Thraustochytrium, microorganisms of thegenus Aurantiochytrium, microorganisms of the genus Schizochytrium,microorganisms of the genus Ulkenia, microorganisms of the genusCrypthecodinium among Alveolata, and mutants thereof tend to producehigher productivity of DHA in particular than other microorganisms. Inthe present specification, these microorganisms are also particularlyreferred to as “DHA highly productive microorganisms.” Exemplary highcontent DHA containing oils may include a DHA highly productivemicrobial oil originating from DHA highly productive microorganisms, abacterium oil originating from a bacterial biomass, a fungus oiloriginating from a fungus biomass, a filamentous fungus oil originatingfrom a filamentous fungus biomass, and the like. Of these, DHA highlyproductive microbial oils tend to have a particularly high DHAconcentration.

Because the classification of microorganisms generally has unconfirmedparts, microorganisms which may produce high content DHA containing oilcan be selected based on criteria other than the abovementionedtaxonomic position. Exemplary criteria other than such a taxonomicposition may include morphology and fatty acid composition. In a casewhere microorganisms are selected based on morphology, themicroorganisms have at least one flagellar basal body and include astructure for accumulating lipids represented by lipid droplets incells, and the like. Upon selection based on the fatty acid composition,examples thereof include tri- or higher unsaturated fatty acid having 20or more carbons. Specific examples thereof include at least one selectedfrom the group consisting of eicosadienoic acid (C20:2, n-9, EDA),dihomo-γ-linolenic acid (C20:3, n−6, DGLA), Mead acid (C20:3, n-9, MA),eicosatetraenoic acid (C20:4, n−3, ETA), arachidonic acid (C20:4, n−6,ARA), eicosapentaenoic acid (C20:5, n−3, EPA), docosatetraenoic acid(C22:4, n−6, DTA), docosapentaenoic acid (C22:5, n−3, _(n-3)DPA), anddocosapentaenoic acid (C22:5, n-6, _(n-6)DPA). Microorganisms may beselected based on both the morphology and fatty acid composition, oronly one thereof.

As long as the microorganisms have the capacity to produce high contentDHA containing oil, the microorganisms may be a wild strain collectedunder a natural environment, or may be a high content DHA containing oilproducing mutant obtained by artificially or incidentally inducingmutation to the wild strain.

As a method for creating mutants, a known mutation inducing method canbe used without being particularly limited. Exemplary methods forcreating mutants may include processes using radiation, mutagens, andthe like. In a case where the mutation inducing process is carried outusing at least one of radiation or a mutagen, the process can be carriedout such that the death ratio of microorganisms is 98%, 99%, or 99.9%.With the mutation inducing process using such a death ratio,microorganisms useful for obtaining the high content DHA containing oilcan be frequently acquired.

In the mutation inducing method involving a process using a mutagen, theselected mutagen is added to microorganisms serving as the target toinduce mutation. The application amount of the mutagen may be theapplication amount capable of inducing mutation and is appropriatelyselected in accordance with the kind of microorganisms, state ofmicroorganisms, load on microorganisms, kind of mutagen, and the like.

While not limited thereto, exemplary selectable mutagens may includeethyl methane sulfonate (EMS), methyl methane sulfonate (MMS),N-ethyl-N-nitrosourea (ENU), triethyl melamine (TEM),N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil,cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan,nitrogen mustard, vincristine, dimethyl nitrosoamine,N-methyl-N′-nitro-nitrosoguanidine (NTG), 2-aminopurine, 7, 12dimethyl-benz(a)anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, busulfan, diepoxy alkanes (diepoxy octane (DEO), diepoxybutane (BEB), and the like),2-methoxy-6-chloro-9[3-(ethyl-2-chloroethyl)aminopropylamino]acridine.dihydrochloride(ICR-170), formaldehyde, and the like. At least one selected from thegroup consisting of these mutagens may be selected.

The state of microorganisms targeted for the process using mutagens,that is, the density, growth stage, and the like can be appropriatelyselected in accordance with the kind and growth state of microorganisms,and the like. The morphology of the microorganisms targeted for theprocess using mutagens may be a growing microbial body (vegetable cells,mycelia, and the like) or zoospores and spores. In terms of efficientlyobtaining a mutant producing the high content DHA containing oil,microorganisms targeted for the process can be microorganisms formingzoospores.

In terms of acquisition efficiency, as microorganisms targeted for themutation inducing process useful for obtaining the high content DHAcontaining oil, at least one microorganism selected from the groupconsisting of microorganisms belonging to the genera Schizochytrium,Thraustochytrium, Aurantiochytrium, and Aplanochytrium is preferable,among which microorganisms of the genus Aurantiochytrium areparticularly preferable, and the at least one microorganism can beprocessed using mutagens to obtain mutants.

Microorganisms useful for obtaining the high content DHA containing oilinclude at least one microorganism selected from the group consisting ofmicroorganisms belonging to the genera Schizochytrium, Thraustochytrium,Aurantiochytrium, and Aplanochytrium, among which microorganisms of thegenus Aurantiochytrium are particularly preferable. The at least onemicroorganism can be processed to obtain mutants using a mutagen, forexample, at least one drug selected from the group consisting of ethylmethane sulfonate (EMS), N-ethyl-N-nitrosourea (ENU), andN-methyl-N′-nitro-nitrosoguanidine (NTG), such that the death ratio ofmicroorganisms is 98%, 99%, or 99.9% with respect to microorganisms inthe growth stage forming the zoospores. These mutants may furtherefficiently produce the high content DHA containing oil.

Alternatively, microorganisms useful for obtaining the high content DHAcontaining oil include a mutant obtained from at least one microorganismselected from the group consisting of Aurantiochytrium limacinum,Aurantiochytrium mangrovei, and the like, by a treatment using amutagen, for example, at least one drug selected from the groupconsisting of ethyl methane sulfonate (EMS), N-ethyl-N-nitrosourea(ENU), and N-methyl-N′-nitro-nitrosoguanidine (NTG), such that the deathratio of microorganisms is 98%, 99%, or 99.9% with respect tomicroorganisms in the growth stage forming the zoospores. These mutantsmay further efficiently produce the high content DHA containing oil.

Exemplary mutants useful for obtaining the high content DHA containingoil may include Aurantiochytrium limacinum NiD2 strain and NiD3 strain,as well as microbial strains having substantially the samemicrobiological properties as these microbial strains. Aurantiochytriumlimacinum NiD2 strain and NiD3 strain, as well as microbial strainshaving substantially the same microbiological properties as thesemicrobial strains, can stably produce the high content DHA containingoil specified in the present specification. Aurantiochytrium limacinumNiD2 strain and NiD3 strain, as well as the NBL-8 strain which is theparent strain thereof, were deposited by the National Institute ofTechnology and Evaluation Patent Organism Depositary Center (#120,2-5-8, Kazusakamatari, Kisarazu-shi, Chiba, Japan) on Oct. 7, 2015(NBL-8 strain: accession number FERM BP-22295, NiD2 strain: accessionnumber FERM BP-22296, and NiD3 strain: accession number FERM BP-22297).

In a case where the high content DHA containing oil is a microbial oil,the high content DHA containing oil can include other componentscharacteristic of the microbial oil, can exhibit a fatty acidcomposition characteristic of microorganisms, or can include othercomponents characteristic of microorganisms and exhibit a peculiar fattyacid composition. Exemplary other components characteristic of themicrobial oil may include phospholipids, free fatty acids, fatty acidesters, monoacyl glycerols, diacyl glycerols, triacyl glycerols, sterolsand sterol esters, carotenoids, xanthophylls, ubiquinones, hydrocarbons,isoprenoid derived compounds, compounds with any of these metabolized,and the like.

Because the high content DHA containing oil as the microbial oil can beobtained without undergoing a chemical synthesis step, the organicsolvent content can be low. The organic solvent in the presentspecification means an organic solvent other than fatty acids and meansa hydrophobic or hydrophilic solvent having at least one carbon atom.Exemplary organic solvents include polar solvents, nonpolar solvents,water miscible solvents, water immiscible solvents, and combinations ofat least two of the solvents. Exemplary organic solvents includesubstituted or unsubstituted, saturated or unsaturated aliphatichydrocarbons, aromatic hydrocarbons, alcohols, ethers, ketones,aldehydes, carboxylic acids, esters, nitriles, amides and the like. Theorganic solvent may be one type of these or a combination of at leasttwo of these.

The total content of the residual organic solvent in the high contentDHA containing oil may be 5000 ppm or less, 3000 ppm or less, 2000 ppmor less, or 1000 ppm or less.

The high content DHA containing oil may have a low content of at leastone selected from the group consisting of methanol, ethanol, acetone,and hexane among the organic solvents. The content of these organicsolvents may each independently be 500 ppm or less, 300 ppm or less, or200 ppm or less. For example, the entire content of methanol, ethanol,acetone, and hexane in the high content DHA containing oil may be 500ppm or less, 300 ppm or less, or 200 ppm or less.

In a case where the high content DHA containing oil is a crude oil of amicrobial oil, the DHA concentration can be 40 wt. % or more, 43 wt. %or more, 50 wt. % or more, 55 wt. % or more, 60 wt. % or more, 65 wt. %or more, or 70 wt. % or more of the total weight of fatty acid in theoil. In a case where the high content DHA containing oil is a crude oilfrom microorganisms, the DHA concentration may be 95 wt. % or less, 90wt. % or less, 85 wt. % or less, or 80 wt. % or less of the total weightof fatty acid in the oil. The upper limit value and lower limit value ofthe DHA concentration in the high content DHA containing oil may be anyof the following combinations. For example, the DHA concentration of thehigh content DHA containing oil can be from 40 wt. % to 95 wt. %, from40 wt. % to 85 wt. %, from 40 wt. % to 80 wt. %, from 60 wt. % to 80 wt.%, from 70 wt. % to 85 wt. %, or from 70 wt. % to 95 wt. % of the totalweight of fatty acid in the oil.

In terms of stability at low temperatures, the high content DHAcontaining oil may be any of the following:

a microbial oil of Aurantiochytrium limacinum which contains DHA at from40 wt. % to 95 wt. % of the total weight of the fatty acid in the oiland has a DSC pour point of 10° C. or lower;

a microbial oil from microorganisms of the genus Aurantiochytrium whichcontains DHA at from 65 wt. % to 80 wt. % of the total weight of fattyacid in the oil and has a DSC pour point of 5° C. or lower;

a crude oil of a microbial oil of Aurantiochytrium limacinum whichcontains DHA at from 40 wt. % to 95 wt. % of the total weight of thefatty acid in the oil and has a DSC pour point of 10° C. or lower;

a crude oil of a microbial oil from microorganisms of the genusAurantiochytrium which contains DHA at from 65 wt. % to 80 wt. % of thetotal weight of the fatty acid in the oil and has a DSC pour point of 5°C. or lower;

a microbial oil from microorganisms of the genus Aurantiochytrium whichcontains DHA at from 40 wt. % to 95 wt. % of the total weight of thefatty acid in the oil and has a DSC pour point from −50° C. to 10° C. orlower; and

a crude oil of a microbial oil of Aurantiochytrium limacinum whichcontains DHA at from 40 wt. % to 80 wt. % of the total weight of thefatty acid in the oil and has a DSC pour point from −50° C. to 5° C. orlower.

The high content DHA containing oil may be contained in the biomass asdescribed later. The high content DHA containing oil may be contained inspecific microbial biomasses, for example, biomasses of Aurantiochytriumlimacinum NiD2 strain (accession number FERM BP-22296), NiD3 strain(accession number FERM BP-22297), and a microbial strain havingsubstantially the same microbiological properties as these strains.

(2) DHA Containing Biomass

The DHA containing biomass according to one embodiment of the presentdisclosure contains the abovementioned high content DHA containing oil,that is, an oil including DHA at a concentration of 40 wt. % or more ofthe total weight of fatty acid in the oil, with the endothermic peaktemperature thereof determined by DSC being 15° C. or lower. In thepresent specification, a biomass containing the high content DHAcontaining oil in one embodiment of the present disclosure is alsosimply referred to as a “high content DHA containing biomass.”

The high content DHA containing biomass can be of any morphology as longas it is an aggregate or mass of microorganisms producible theabovementioned high content DHA containing oil. For example, the highcontent DHA containing biomass may be an aggregate or mass ofmicroorganisms in a culture liquid, an aggregate or mass ofmicroorganisms after being recovered from the culture liquid, or anaggregate or mass of microorganisms undergoing a drying process afterbeing recovered from the culture liquid. In the event of particularlyindicating the high content DHA containing biomass via the dryingprocess, it is also referred to as “a high content DHA containing driedbiomass” in the present specification. The high content DHA containingdried biomass is in the dried form, allowing it to be easily handled.

In the high content DHA containing biomass or the high content DHAcontaining dried biomass, the high content DHA containing oil may bepresent in individual cells which make up the biomass and may bereleased outside the cells in a case where it can be recovered from thecultured biomass as part of the biomass.

Regarding microorganisms which make up the high content DHA containingbiomass, subjects described in regard to microorganisms in the highcontent DHA containing oil except for the subjects specified below aredirectly applied as subjects replaced with microorganisms in thebiomass.

In terms of the growth of microorganisms and lipid accumulation,exemplary microorganisms in the high content DHA containing biomass mayinclude microorganisms belonging to Stramenopiles, preferablyThraustochytrid, as well as microorganisms belonging to Alveolata,preferably the genus Crypthecodinium. At least one microorganismselected from the group consisting of microorganisms of the genusSchizochytrium, microorganisms of the genus Crypthecodinium, andmicroorganisms of the genus Aurantiochytrium can be further preferablyselected. Microorganisms of the genus Aurantiochytrium can beparticularly selected. As long as these microorganisms have the capacityto produce the DHA containing oil, the microorganisms may be wildstrains collected under a natural environment or may be mutants obtainedby artificially or incidentally inducing mutations in the wild strains.Regarding the mutants which may form the high content DHA containingbiomass, subjects described regarding the mutants in the high contentDHA containing oil are replaced with mutants in the biomass and directlyapplied. Exemplary mutants of microorganisms in the high content DHAcontaining biomass may include Aurantiochytrium limacinum NiD2 and NiD3strains, along with microbial strains having substantially the samemicrobiological properties as these microbial strains.

NiD2 and NiD3 strains, along with microbial strains having substantiallythe same microbiological properties as these microbial strains, canproduce high content DHA containing oil containing DHA at aconcentration of 40 wt. % or more of the total weight of the fatty acidwith a DSC pour point of 15° C. or lower in a biomass including thesemicrobial strains or in the cell thereof. The high content DHAcontaining oil produced by these microbial strains exhibits a tendencyin which the content of _(n-6)DPA in the oil is higher than the contentof that in the oil produced by the parent strains of these microbialstrains, a tendency in which the content of palmitic acid is lower thanthe content of that in the oil produced by the parent strains of thesemicrobial strains, or a tendency combining these.

The high content DHA containing biomass may efficiently produce DHA. Theaccumulative amount of DHA of the high content DHA containing biomassmeans the amount of DHA produced by the biomass in 1 liter of cultureproduct and is determined as the ratio (weight ratio) of the DHA amountto the culture liquid per 1 liter of the dried biomass weight. The DHAaccumulative amount of the high content DHA containing biomass, forexample, can be 18 wt. % or more, 20 wt. % or more, 23 wt. % or more, 28wt. % or more, 30 wt. % or more, 33 wt. % or more, 38 wt. % or more, 40wt. % or more, 48 wt. % or more, 50 wt. % or more, or 53 wt. % or moreof the dried biomass weight. Because the high content DHA containingbiomass includes the high content DHA containing oil having such a DHAconcentration, for example, the high content DHA containing oil can beefficiently obtained from the biomass by generally used extractionmethods. Moreover, by taking in the high content DHA containing biomassor the high content DHA containing dried biomass as is, the high contentDHA containing oil can be efficiently taken in.

(3) Manufacturing Method

The manufacturing method of the high content DHA containing oil caninclude: culturing organisms such as a microbial strain of the genusAurantiochytrium, which may produce an oil which contains DHA at aconcentration of 40 wt. % or more of the total weight of fatty acid inthe oil with the endothermic peak temperature thereof determined bydifferential scanning calorimetry (DSC) being 15° C. or lower, that is,a high content DHA containing oil, to obtain a biomass (hereinafter,also referred to as a culture step); recovering a cultured biomass(hereinafter, also referred to as a recovery step); extracting oil fromthe recovered biomass (hereinafter, also referred to as the extractionstep), and can further include other steps as required.

The manufacturing method of the biomass according to one aspect of thepresent disclosure can include the abovementioned culture step andrecovery step, in addition to further including other steps as required.

The manufacturing method of the dried biomass according to one aspect ofthe present disclosure can include the abovementioned culture step andrecovery step, and drying the biomass obtained by recovery (hereinafter,also referred to as a drying step), in addition to further includingother steps as required.

In the culture step, organisms which may produce the high content DHAcontaining oil are cultured. The organisms used in the culture step arenot limited as long as they are organisms which may produce the highcontent DHA containing oil. The culture conditions, culture medium,culture apparatus, and the like, which are generally applied toorganisms which may produce the high content DHA containing oil, can bedirectly applied.

In a case where organisms which may produce the high content DI-IAcontaining oil are microorganisms, the spores or mycelia of themicrobial strain or preculture liquid obtained by culturingmicroorganisms in advance are inoculated to a liquid or solid culturemedium and be cultured. Exemplary culture media may include: carbonsources such as glucose, fructose, xylose, sucrose, maltose, starch,soluble starch, molasses, glycerol, and mannitol; nitrogen sources suchas yeast extract, malt extract, meat extract, fish meat extract, cornsteep liquor, peptone, polypeptone, soybean powder, defatted soybeanpowder, casamino acid, urea, sodium glutamate, ammonium acetate,ammonium sulfate, ammonium nitrate, ammonium chloride, and sodiumnitrate; inorganic salts such as sodium chloride, potassium chloride,magnesium chloride, magnesium sulfate, calcium chloride, potassiumdihydrogen phosphate, and dipotassium hydrogen phosphate; vitamins; andculture media obtained by appropriately combining necessary components.The culture medium includes sodium chloride, potassium chloride,magnesium chloride, magnesium sulfate, calcium chloride, and the like,and therefore can be applied to the culture medium of marinemicroorganisms. In terms of the growth potential of a wide variety ofmicroorganisms which may produce high content DHA containing oil, forexample, yeast extract glucose agar culture medium (GY medium) can beused as the culture medium. An aqueous medium used as the base materialfor the liquid culture medium is basically water, with distilled wateror purified water capable of being used. After preparing the culturemedium, the pH is adjusted to the range from 3.0 to 9.0, after which theculture medium is sterilized by an autoclave, and the like. Culturingcan be carried out at a culture temperature from 10° C. to 40° C. for aperiod of 1 to 14 days by ventilation stirring culture, shaking culture,or stationary culture.

The spores or mycelia of the microbial strain or the preculture liquidobtained by preculturing microorganisms in advance can be inoculated toa liquid or solid culture medium and be cultured. Exemplary culturemedia may include: carbon sources such as glucose, fructose, xylose,sucrose, maltose, starch, soluble starch, molasses, glycerol, andmannitol; nitrogen sources including natural nitrogen sources such asyeast extract, malt extract, meat extract, fish meat extract, corn steepliquor, peptone, polypeptone, soybean powder, defatted soybean powder,and casamino acid, organic nitrogen sources such as urea, and inorganicnitrogen sources such as sodium glutamate, ammonium acetate, ammoniumsulfate, ammonium nitrate, ammonium chloride, and sodium nitrate;inorganic salts such as phosphate, magnesium sulfate, iron sulfate, andcopper sulfate, and trace nutrient sources such as vitamins; and culturemedia obtained by appropriately combining necessary components. Such aculture medium can be applied to soil derived microorganisms. An aqueousmedium used as the base material for the liquid culture medium isbasically water, with distilled water or purified water capable of beingused.

In terms of maintenance or promotion of the growth of microorganisms,the nitrogen source in the culture medium can be from 0.01 wt. % to 10wt. %, preferably from 0.1 wt. % to 4 wt. %. An appropriate amount ofthe nitrogen source can be added into the culture medium along withgrowth of microorganisms, with no limitation on the number of times. Inthe case of adding a nitrogen source, the amount of the nitrogen sourceto be once added can be appropriately set in terms of maintenance orpromotion of the growth of microorganisms.

In terms of maintenance or promotion of the growth of microorganisms,the carbon source in the culture medium can be from 0.1 wt. % to 30 wt.%, preferably from 1 wt. % to 15 wt. %. An appropriate amount of thecarbon source can be added into the culture medium along with the growthof microorganisms, with no limitation on the number of times. In thecase of adding a carbon source, the amount of the carbon source to beonce added can be appropriately set in terms of maintenance or promotionof the growth of microorganisms.

In a case where the following (i) to (iv) microorganisms are selected,the addition amount of sodium salt, and the addition amount of vitamins,can be selected within a specific range. In a case where the additionamount of the sodium salt and the addition amount of the vitamins areselected within a specific range, the DHA production efficiency tends tobe improved due to these marine microorganisms:

(i) marine microorganisms;

(ii) as DHA highly productive microorganisms, at least one microorganismselected from the group consisting of microorganisms of the genusThraustochytrium, microorganisms of the genus Aurantiochytrium,microorganisms of the genus Schizochytrium, microorganisms of the genusUlkenia, the genus Crypthecodinium, and the mutants thereof;

(iii) at least one microorganism selected from the group consisting ofmicroorganisms of the genus Aurantiochytrium such as Aurantiochytriumlimacinum and Aurantiochytrium mangrovei, and the mutants thereof; or

(iv) at least one microorganism selected from the group consisting ofAurantiochytrium limacinum NiD2 and NiD3, and microbial strains havingsubstantially the same microbiological properties as these microbialstrains.

That is, in a case where these marine microorganisms, for example, DHAhighly productive microorganisms, are selected, the addition amount ofsodium salt to the culture medium can be selected in terms ofmaintenance or promotion with respect to, for example, the growth ofmicroorganisms, synthesis of fatty acids, and synthesis of DHA, and canbe from 0.001 wt. % to 10 wt. %, preferably from 0.1 wt. % to 5 wt. %,more preferably from 1.0 wt. % to 3.0 wt. %. In a case where theconcentration of the sodium salt in the culture medium is presumablydecreased along with the addition of the nitrogen source or the carbonsource, the sodium salt concentration in the culture medium can beincreased in the concentration range in advance so as not to inhibit thegrowth of microorganisms. An appropriate amount of sodium salt can beadded to the culture medium, with no limitation on the number of times.In the case of adding sodium salt, the amount of sodium salt to be onceadded can be appropriately set in terms of maintenance or promotion ofthe growth of microorganisms.

In a case where the marine microorganisms, for example, DHA highlyproductive microorganisms, are selected, the addition amount of vitaminsto the culture medium can be selected in terms of maintenance orpromotion of the growth of microorganisms, and production suppression ofodd chain fatty acids, and can be from 0.01 ppm to 50 ppm, preferablyfrom 0.1 ppm to 10 ppm as the total amount of vitamins. Vitamins in theform of a prepared concentrated solution may be added into the culturemedium. In a case where the vitamin concentration in the culture mediumis presumably reduced along with the addition of the nitrogen source orthe carbon source or other culture medium components, the amount ofvitamins in the culture medium can be increased in the concentrationrange in advance so as not to inhibit the growth of microorganisms. Anappropriate amount of vitamins can be added to the culture medium, withno limitation on the number of times. In the case of adding vitamins,the amount of vitamins to be once added can be appropriately set interms of maintenance or promotion of the growth of microorganisms.

In a case where the marine microorganisms, for example, DHA highlyproductive microorganisms, are selected, the addition amount of metalsalts in the culture medium can be selected in terms of maintenance orpromotion with respect to, for example, the growth of microorganisms,synthesis of fatty acids, synthesis of DHA, and the total amount ofmetal salts can be from 0.0001 wt. % to 5 wt. %, preferably from 0.001wt. % to 2 wt. %. Metal salts in the form of a prepared concentratedsolution may be added to the culture medium. In a case where the metalsalt concentration in the culture medium is presumably decreased alongwith the addition of the nitrogen source, carbon source, or otherculture medium components, the amount of metal salts in the culturemedium can be increased in the concentration range in advance so as notto inhibit the growth of microorganisms. An appropriate amount of metalsalts can be added to the culture medium, with no limitation on thenumber of times. In the case of adding metal salts, the amount of metalsalts to be once added can be appropriately set in terms of maintenanceor promotion of the growth of microorganisms.

In a case where the marine microorganisms, for example, DHA highlyproductive microorganisms, are selected, the pH of the culture mediumcan be selected in terms of maintenance or promotion with respect to,for example, the growth of microorganisms, synthesis of fatty acids, andsynthesis of DHA, and can be from 3.0 to 9.5, preferably from 3.5 to9.5, more preferably from 4.5 to 9.0. The pH of the culture medium canbe appropriately controlled by a pH adjustor such as sodium hydroxide orsulfuric acid in a case where components in the culture medium aremetabolized with microorganisms so as to increase or decrease the pH.

In terms of the DHA production efficiency, the culture temperature canbe, for example, 40° C. or lower, 35° C. or lower, 30° C. or lower, 28°C. or lower, or 25° C. or lower, while in terms of the growth ofmicroorganisms, it can be 8° C. or higher, 10° C. or higher, or 15° C.or higher. The upper limit value or lower limit value of the culturetemperature may be any of the following combinations, and can be, forexample, from 8° C. to 40° C., from 8° C. to 28° C., from 10° C. to 40°C., from 10° C. to 25° C., or from 15° C. to 25° C. In the case ofculturing in such a temperature range, the DHA concentration in the highcontent DHA containing oil can be increased.

No particular limitation is placed on the culture vessel used forculturing, and any device that is ordinarily used for the culturing ofmicrobes can be used. The culture vessel may be appropriately selectedaccording to the scale of culturing. Examples thereof may include aculture vessel which enables liquid culturing at a scale of from 1 L to50 L. The culture vessel can include a stirring or penetration function.In the case of liquid culturing at a scale from 1 L to 50 L, astirred-type culture vessel is preferably used as the culture vessel inorder to obtain the target high content DHA containing oil at a higherconcentration. The stirred-type culture vessel preferably has discturbine-type agitator blades in at least one stage, and a stirred-typeculture vessel further preferably has disc turbine type agitator bladesin two stages.

While not particularly limited thereto, the culture period in theculture step differs depending on the kind and growth state ofmicroorganisms, the accumulation efficiency of fatty acids, and thelike, and can be completed when the amount of the dried biomass per 1liter of the culture liquid reaches from 20 g to 200 g. The cultureperiod in the culture step can be generally from 1 to 14 days, or from 3to 10 days. The airflow rate in the ventilation culture can generally befrom 0.2 vvm to 2.0 vvm, preferably from 0.5 vvm to 1.0 vvm. Moreover,the internal gauge pressure can generally be from 0.01 MPa to 0.2 MPa,preferably from 0.02 MPa to 0.1 MPa.

In the recovery step, the cultured biomass is recovered. In accordancewith the kind of organisms and the kind of culture medium, the culturedbiomass can be recovered from the culture medium by methods known in theart. In the recovery step, a biomass including the high content DHAcontaining oil is obtained. For example, in a case where a culture iscarried out using a liquid culture, the recovery process can be carriedout in general using a solid-liquid separation means such ascentrifugation and filtration after the completion of culturing. In acase where a solid culture medium is used for culturing, the solidculture medium and the biomass may be crushed using a homogenizer andthe like without separation of the biomass from the culture medium, andthe crushed material obtained may be supplied to the extraction step.

In order to obtain the dried biomass, the drying step in which thebiomass obtained in the recovery step if dried may be included prior tothe extraction step. The drying process can be carried out by methodsknown in the art, such as freeze drying, air drying, or heat drying.Whether a biomass has been obtained via the drying process can beconfirmed by measuring the water content. For example, the water contentof a dry microbial biomass can be 10% or less. The water content can bemeasured based on the “Normal pressure heat drying method” described inthe “Shin Shokuhin bunsekiho” (January, 1997) edited by the JapaneseSociety for Food Science and Technology, Food Analysis Method EditingCommittee.

In the extraction step, an oil is extracted from the biomass after therecovery step to obtain an extracted oil. Generally, a standardextraction method using an organic solvent can be applied to theextraction process. In a case where the recovered biomass is driedbiomass, the extraction process may involve extraction usingsupercritical carbon dioxide or may be an extraction process using anorganic solvent under nitrogen air flow. Exemplary organic solvents thatcan be used in the extraction process may include: ethers such asdimethylether and diethylether; hydrocarbons having 10 or less carbonssuch as petroleum ether, hexane, and heptane; alcohols such as methanoland ethanol; chloroform; dichloromethane; fatty acids such as octanoicacid or alkyl esters thereof; and oils and fats such as vegetable oil.As the extraction process, extraction by alternating extraction usingmethanol and petroleum ether or extraction using a single layer typesolvent of chloroform-methanol-water can be applied. In this case, morefavorable results from the extraction process can be obtained. Anextracted oil can be obtained by distilling off the organic solvent fromthe extract under reduced pressure. In a case where triacyl glycerol iscollected or the extracted oil is extracted from microorganisms, hexaneis generally used as the organic solvent. As mentioned above, crude oilrefers to the extracted oil obtained immediately after the extractionprocess.

The high content DHA containing oil can be obtained from the extractedoil. In a case where the high content DHA containing oil is a crude oil,the high content DHA containing oil can be obtained in the extractionstep. Because the extracted oil obtained in the extraction step, thatis, the crude oil contains 40 wt. % or more DHA and the DSC pour pointthereof is 15° C. or lower, it is relatively stable against changes intemperature, and shows an excellent stability at low temperatures andexcellent handleability. For example, in a case where crude oil isapplied to the extraction step or the subsequent process, the generationof clogging inside the pipe can be suppressed; and in a case where thecrude oil is blended to prepare a composition, an increase in theviscosity of the composition for blending the crude oil can besuppressed to facilitate a preparation thereof.

In a case where a wet microbial biomass is used in the extractionprocess, the wet microbial biomass may be compressed and a solvent maybe used. Exemplary solvents which can be used may include a solventcompatible with water such as methanol or ethanol, or a mixed solventformed from a solvent compatible with water and water and/or anothersolvent. The remainder of the procedure is similar to that describedabove.

In a case where the high content DHA containing oil is crude oil, thehigh content DHA containing oil can have at least one, at least two, atleast three, at least four, at least five, or all of the followingproperties. In a case where it has two or more properties, anycombination of the below-mentioned (1) to (6) can be used.

(1) The acid value (AV) may be higher than 0.5 mg KOH/g, higher than 1.0mg KOH/g, or higher than 1.5 mg KOH/g.

(2) The phospholipid concentration may be 3 wt. % or more, 5 wt. % ormore, or 10 wt. % or more.

(3) The glycolipid concentration may be 3 wt. % or more, 5 wt. % ormore, or 10 wt. % or more.

(4) The triglyceride concentration may be 90 wt. % or more, 92 wt. % ormore, or 94 wt. % or more.

(5) The DHA concentration in triglycerides may be 50 wt. % or more, 60wt. % or more, 70 wt. % or more, or 80 wt. % or more.

(6) The concentration of unsaponifiable substance may be more than 4.5wt. %.

The acid value (AV) was measured in accordance with the standard methodsfor the analysis of fats, oils and related materials (2013 Edition)2.3.1-2013 acid value (edited by the Japan Oil Chemists' Society).

The phospholipid concentration was measured in accordance with thestandard methods for the analysis of fats, oils and related materials(2013 Edition) 2.4.11-2013 phospholipid (edited by the Japan OilChemists' Society).

The glycolipid concentration was determined as follows. That is, inaccordance with the method described in Lipid analysis: isolation,separation, identification, and structural analysis of lipids, Christie,W. W. Oily Press: Bridgwater, England, 2003; pp 69: Chapter 4 “Analysisof simple lipid classes, A. Preliminary fractionation of lipidextracts”, the fraction of glycolipids was obtained. Regarding silicagel, Sep-Pak (registered trademark) Plus Silica Cartridges (WatersCorporation) having a particle diameter from 55 μm to 105 μm, as well asa capacity of 690 mg (Sorbent weight), was used. The lipid amountobtained after removing acetone in the obtained fraction along withother solvents, if present, was measured, with the weight percentage ofthe obtained lipid amount to the total lipid amount serving as theglycolipid concentration.

Based on the specific method described in the quantitative determinationof unsaponifiable substance of the standard methods for the analysis offats, oils and related materials by the Japan Oil Chemists' Society, theconcentration of unsaponifiable substance was obtained by saponifyingoil and fat, removing the mixed fatty acid amount from the extractedmaterial in a solvent used for quantitative determination, andrepresenting it as the percentage in a sample. Note that, for example,the amount of unsaponifiable substance such as tocopherol added afterrefining is subtracted. The outline of the abovementioned specificmethod is as follows (refer to Oil Chemistry, 13, 489(1996)).

A sample of approximately 5 g is taken in a flask, after which 50 ml of1N-ethanol-potassium is added thereto, gently boiled for 1 hour, andsaponified. Once saponification is complete, heating is stopped, and theflask for saponification is washed with 100 ml of warm water, while aliquid obtained after saponification is transferred into a separatoryfunnel, after which 50 ml of water is added thereto, and cooled until itreaches room temperature. Next, 100 ml of ethyl ether is added to theseparatory funnel while the flask for saponification is washed. Theseparatory funnel is hermetically sealed, shaken hard for 1 minute,mixed, and left to stand until it is clearly divided into two layers.The divided lower layer is transferred into a second separatory funnel,50 ml of ethyl ether is added thereto, shaken and mixed as in the firstseparatory funnel, then left to stand. In a case where it is dividedinto two layers, the lower layer is transferred to a third separatoryfunnel and extraction is similarly repeated using 50 ml of ethyl ether.

Each separatory funnel of the ethyl ether layer in the second and thirdseparatory funnels is washed with a small amount of ethyl ether andtransferred to the first separatory funnel, after which 30 ml of wateris added thereto, shaken, and mixed, subsequently left to stand, anddivided into two layers to remove the lower layer. Furthermore, eachtime, 30 ml of water is added, shaken, and mixed, left to stand, andseparated, after which this is repeated, then the separated water iswashed until it is no longer colored with a phenolphthalein indicator.The washed ethyl ether extracted liquid is subjected to a dehydrationprocess with sodium sulfate (anhydrous) as required, filtrated withdried filter paper, then transferred to a distillation flask. Severalcontainers, filter paper, and the like used for the extraction are allwashed with a small amount of ethyl ether, and a washing liquid is addedto the distillation flask. Ethyl ether in the distillation flask isdistilled and removed, then cooled in a case where the liquid amountthereof reaches approximately 50 ml. Subsequently, the flask is washedwith a small amount of ethyl ether, while the concentrated ethyl etherextract is transferred to a 100 ml round bottom flask with the weightthereof precisely measured in advance.

Most of the ethyl ether in the round bottom flask is distilled andremoved, 3 ml of the acetone is subsequently added, the majority thereofis distilled and removed as before, for example, the extract is heatedunder slightly reduced pressure of approximately 200 mmHg at 70 to 80°C. for 30 minutes, and the round bottom flask is transferred to a vacuumdesiccator, left to stand for 30 minutes, and cooled. The weight of theround bottom flask is precisely measured to determine the weight of theextract. 2 ml of ethyl ether and 10 ml of neutral ethanol are added tothe round bottom flask and sufficiently shaken and mixed to dissolve theextract, then the amount of mixed fatty acids is determined in anN/10-ethanol-potassium standard solution using the phenolphthaleinindicator. Here, the end point is reached when the slightly red color ofthe titration indicator is maintained for 30 seconds.Unsaponifiable substance concentration (%)=A−(B×F×0.0282)/C×100mixed fatty acid(as oleic acid, g)=B×F×0.0282wherein

A=weight (g) of the extract

B=usage amount (ml) of the N/10-ethanol-potassium standard solution

C=amount (g) of the sample collected

F=potency of the N/10-ethanol-potassium standard solution

The triglyceride concentration was measured in accordance with AOCSRecommended Practice Cd 11c-93 (1997), SAMPLING AND ANALYSIS OFCOMMERCIAL FATS AND OILS, (American Oil Chemists' Society).

In a case where the high content DHA containing oil is a refined oil,the manufacturing method of the high content DHA containing oil caninclude, after the extraction step, the refining process in which theobtained extracted oil, that is, crude oil is subjected to the refiningstep including a degumming step, deacidification step, decoloring step,and deodorizing step. The high content DHA containing oil as the refinedoil can be obtained in the refining process. In a case where theextracted oil derived from a microbial biomass is subjected to therefining process, a triacyl glycerol concentrate having a higher triacylglycerol concentration than the crude oil is mainly obtained.

In the refining step, degumming, deacidification, decoloring, anddeodorizing are performed on the crude oil with methods used for thepurification of vegetable oil, fish oil, and the like using methodsknown to those skilled in the art. The degumming process is carried out,for example, by the washing process, and the like. The deacidificationprocess is carried out, for example, by the distillation process, andthe like. The decoloring process is carried out, for example, by thedecoloring process using activated white clay, activated carbon, silicagel, and the like. The deodorizing process is carried out, for example,by steam distillation, and the like. Because the refined oil obtained inthe refining step contains 40 wt. % or more DHA and the DSC pour pointthereof is 15° C. or lower, it is relatively stable against changes intemperature and shows excellent stability at low temperatures.Therefore, as with crude oil, in the refining step or the subsequentprocess, the generation of clogging inside a pipe can be suppressed,resulting in excellent handleability.

The refined oil obtained after the refining step may have a triglycerideconcentration of 95 wt. % or more and satisfy at least one conditionselected from the group consisting of the following (P1) to (P4), or maysatisfy at least one condition selected from the group consisting of thefollowing (P1) to (P4) and have a DHA concentration in triglyceride of50 wt. % or more, 60 wt. % or more, 70 wt. % or more, or 80 wt. % ormore.

(P1) the acid value (AV) is 0.5 mg KOH/g or lower,

(P2) the phospholipid concentration is less than 3 wt. %,

(P3) the glycolipid concentration is less than 3 wt. %, and

(P4) the concentration of unsaponifiable substance is 4.5 wt. % or less.

The manufacturing method of the high content DHA containing oil caninclude a processing step in which the crude oil or the refined oil issubjected to processing such as hydrolysis, alkyl esterification and thelike after the extraction step, or as the case may be, after theextraction step and the refining step. In the processing step, acomposition containing free fatty acids, a composition containing fattyacid lower alkyl esters, and the like can be obtained. In particular, ina case where the manufacturing method of the high content DHA containingoil includes the refining step and processing step in this order afterthe extraction step, free fatty acids, fatty acid lower alkyl esters,and the like, derived from a triacyl glycerol concentrate obtained inthe refining step, can be obtained. The alkyl esterification process andhydrolysis process in the processing step can be carried out underconditions known to those skilled in the art.

Lower alcohols are used in the alkyl esterification process. Exemplarylower alcohols may include alcohols having from 1 to 3 carbons such asmethanol, ethanol, and propanol. For example, the fatty acid methylesters are obtained by processing the extracted oil at room temperaturefor 1 to 24 hours using from 5% to 10% anhydrous methanol/hydrochloricacid, from 10% to 50% BF3/methanol, and the like. The ethyl esters ofthe fatty acids are obtained by processing the extracted oil for 15 to60 minutes at 25° C. to 100° C. using from 1% to 20% sulfuric acidethanol, and the like. The methyl esters or ethyl esters may beextracted from the reaction liquid using an organic solvent such ashexane, diethylether, or ethyl acetate. The extract liquid is driedusing anhydrous sodium sulfate or the like, after which the organicsolvent is removed by distillation to obtain a composition mainlycomposed of fatty acid alkyl esters.

The hydrolysis process may be carried out under conditions known tothose skilled in the art, for example, such that extracted oil issubjected to an alkaline decomposition process with 5% sodium hydroxideat room temperature for 2 to 3 hours, after which a compositioncontaining free fatty acids can be obtained from the obtaineddecomposition liquid using a method regularly used for the extraction orrefining of fatty acids.

In order to obtain free fatty acids, both the alkyl esterificationprocess and the hydrolysis process may be carried out. In a case wherethe alkyl esterification process and the hydrolysis process arecontinuously carried out, free fatty acids of a higher purity can beobtained. In order to obtain free fatty acids from fatty acid alkylesters, for example, after hydrolysis using an alkaline catalyst, theextraction process may be performed using an organic solvent such asether and ethyl acetate.

The manufacturing method of the high content DHA containing oil caninclude a concentration process after the refining step or processingstep, in order to reduce the concentration of fatty acids in which fattyacids other than DHA is constituent fatty acids and to increase the DHAconcentration. For concentration process, distillation, rectification,column chromatography, low temperature crystallization method, ureaclathrate method, liquid-liquid countercurrent distributionchromatography, and the like may be used alone or in combination of twoor more. A combination of distillation or rectification, along withcolumn chromatography or liquid-liquid countercurrent distributionchromatography is preferably used. Reverse phase distribution typecolumn chromatography is preferable for column chromatography. When thestep of concentrating or isolating DHA is performed, the content of DHA,which may be finally contained in the high content DHA containing oil,in the fatty acids can be increased and the content of fatty acids otherthan DHA in the fatty acids can be decreased.

For example, for the case in which rectification is used, therectification step is preferably carried out by distillation under areduced pressure at the upper part of the distillation column of lessthan or equal to 10 mmHg (1333 Pa), and temperature of the column bottomin the range from 165° C. to 210° C., preferably from 170° C. to 195°C., from the perspective of suppressing the denaturation of fatty acidsby heating and increasing the efficiency of rectification. The pressureof the upper part of the distillation column is preferably as low aspossible, preferably 0.1 mmHg (13.33 Pa) or lower. No particularlimitation is placed on the temperature at the upper part of the column,and for example, this temperature may be set to 160° C. or lower. In therectification step, a composition having an even higher content of DHAmay be obtained.

The reverse phase column chromatography may be a type of reverse phasecolumn chromatography that is known in the art, and high-performanceliquid chromatography (HPLC) using a substrate modified byoctadecylsilyl groups (ODS) as a stationary phase is particularlypreferable.

By carrying out such a concentration process, a DHA concentrated oilhaving a DHA concentration, for example, from 90 wt. % to 98 wt. %, from95 wt. % to 98 wt. %, from 96 wt. % to 98 wt. %, or from 97 wt. % to 98wt. % of the total weight of fatty acid in the oil, can be obtained.

(4) Use

The high content DHA containing oil and high content DHA containingbiomass DHA can be applied for uses in which various functions arerequired to efficiently obtain. Exemplary products containing the highcontent DHA containing oil and high content DHA containing biomass mayinclude food products, supplements, medicaments, cosmetics, animal feed,and the like. The high content DHA containing oil and high content DHAcontaining biomass can be used in the manufacturing method of theseproducts. In particular, exemplary uses as the high content DHAcontaining oil and high content DHA containing biomass include foodproducts, supplements, medicaments, cosmetics, animal feed, and the likewhich include DHA as an effective component, and are expected to beeffective in terms of the prevention of lifestyle related diseases suchas arteriosclerosis, cerebral infarction, myocardial infarction,thrombosis, and hyperlipemia, improvement of metabolic syndrome,antiallergies, antiinflammation, and anticancer, improved brainfunction, and improved stress. Exemplary medicaments include externalmedicines for skin and oral preparations.

The use form of the high content DHA containing oil, high content DHAcontaining biomass, and high content DHA containing dried biomass is notparticularly limited and can be used as liquid components or solidcomponents. For example, in a case where the high content DHA containingoil, high content DHA containing biomass, or high content DHA containingdried biomass is applied to products such as food products, supplements,medicaments, cosmetics, and animal feed, the high content DHA containingoil, high content DHA containing biomass, or high content DHA containingdried biomass itself may be combined with other components forcommercialization or can be subjected to additional processes before orafter being combined with other components for commercialization.Exemplary additional processes may include powderization, pelletization,capsulation, tableting, and the like.

In a case where the high content DHA containing oil, high content DHAcontaining biomass, and high content DHA containing dried biomass areused as medicaments, medicaments include the high content DHA containingoil, high content DHA containing biomass, and high content DHAcontaining dried biomass, pharmaceutically acceptable carriers and,optionally, other components. The dosage form may be any form which isconvenient for oral administration or parenteral administration.Exemplary dosage forms include injections, transfusions, powders,granules, tablets, capsules, enteric coated tablets, troches, peroralliquid preparations, suspensions, emulsions, syrups, liquids forexternal use, fomentations, nasal preparations, ear drops, eye drops,inhalants, ointments, lotions, and suppositories. These may be usedindividually or in combination depending on the symptoms.

By standard methods, these various types of preparations, depending onthe purpose, may be formulated by adding, to the principle agent,previously known adjutants commonly used in the field of drugpreparation technology, as exemplified by excipients, binders,preservatives, stabilizers, disintegrants, lubricants, flavoring agents,and the like. Furthermore, in the case of oral administration to adults,typically, the dosage for administration can be appropriately adjustedwithin a range from 0.01 mg to 10 g, preferably from 0.1 mg to 2 g, andmore preferably from 1 mg to 200 mg, per day as the total amount of DHAas a structured lipid. In the case of parenteral administration, thedosage for administration can be appropriately adjusted within a rangefrom 0.001 mg to 1 g, preferably from 0.01 mg to 200 mg, and morepreferably from 0.1 mg to 100 mg, per day as the total amount of DHA asa structured lipid. These dosages differ depending on the purpose of theadministration, along with the conditions of the person subjected toadministration, for example, sex, age, weight, and the like.

In the present specification, the features described in the embodimentsrelated to each aspect of the disclosure may be combined as desired toform new embodiments, and it is to be understood that such newembodiments may be included in each of the aspects of the presentdisclosure.

EXAMPLES

The present disclosure is described below in detail using examples.However, the present disclosure is not limited in any manner by theseexamples. Unless otherwise specified, “part” or “%” is indicated on amass basis.

Unless otherwise specified, in the following examples, “cell(s)” or“microbial body(ies)” mean an aggregation of cells or microbial bodies,corresponding to the biomass in the present specification.

Example 1

Isolation and Identification of the NBL-8 Strain

Mud was collected from the brackish water region of Ishigaki Island. Theobtained mud was appropriately diluted with artificial seawater, appliedto an agar culture medium for Thraustochytrid including 1 mg/ml ofampicillin, and cultured at 26° C. On the third day of culturing,multiple microbial strains for forming colonies were collected in theculture medium, one of which was named the NBL-8 strain.

The NBL-8 strain has a life history of vegetable cells and zoospores ina liquid culture medium for Thraustochytrid, wherein the vegetable cellsof the NBL-8 strain do not exhibit motility and are proliferated mainlyby continuous binary fission. The NBL-8 strain forms zoosporangia andforms multiple zoospores in the zoosporangia. Subsequently, thezoospores are released outside the zoosporangia to serve as zoospores.The zoospores are grounded after a certain period of swimming to serveas vegetable cells. Such a life history is characteristic of the kingdomChromista, the class Labyrinthulea, the family Thraustochytriaceae, thegenus Aurantiochytrium, and the species limacinum (refer to Honda et.al., Mycological Research. 102(4):439-448(1998); Yokoyama and Honda,Mycoscience. 48(4):199-211(2007)). From the analytical results of the16S rRNA gene sequence as well, the NBL-8 strain is recognized asAurantiochytrium limacinum.

Note that the name of Aurantiochytrium limacinum during the report thatit is separated is Schizochytrium limacinum. The classification of thefamily Thraustochytriaceae was reviewed by Honda et. al. in 2007, andrenamed Aurantiochytrium (refer to the abovementioned document).

Each composition of artificial seawater, an agar culture medium forThraustochytrid, and a liquid culture medium for Thraustochytrid, whichwas used to isolate the NBL-8 strain, is indicated in Tables 1 to 3. Avitamin solution obtained by dissolving each composition, sterilizing itwith a filter, and storing it at −20° C. was used. A metal salt solutionobtained by dissolving each composition, sterilizing it, and storing itat 4° C. was used. These solutions mentioned above were also used in thefollowing examples.

Each composition of the vitamin solution (*) and the metal salt solution(**) in Tables 2 and 3 is indicated in Tables 4 and 5.

TABLE 1 Artificial seawater (1 liter) NaCl  15 g KCl 0.35 g  MgCl₂•6H₂O5.4 g MgSO₄•7H₂O 2.7 g CaCl₂•2H₂O 0.5 g Deionized water Residual

TABLE 2 Agar culture medium for Thraustochytrid (1 liter) Glucose 30 gYeast extract 20 g NaCl 15 g KCl 0.35 g MgCl₂•6H₂O 5.4 g MgSO₄•7H₂O 2.7g CaCl₂•2H₂O 0.5 g Vitamin solution * 1.0 mL Metal salt solution ** 2.0mL Agar powder 15 g Deionized water Residual

TABLE 3 Liquid culture medium for Thraustochytrid (1 liter) Glucose 30 gYeast extract 20 g NaCl 15 g KCl 0.35 g MgCl₂•6H₂O 5.4 g MgSO₄•7H₂O 2.7g CaCl₂•2H₂O 0.5 g Vitamin solution * 1.0 mL Metal salt solution ** 2.0mL Deionized water Residual

TABLE 4 Vitamin solution (1 liter) Thiamine 200 mg  Riboflavin 1.0 mgCyanocobalamin 1.0 mg Deionized water Residual

TABLE 5 Metal salt solution (1 liter) Na₂EDTA  30 g FeCl₃•6H₂O 1.43 gH₃BO₃ 34.2 g MnCl₂•4H₂O  4.3 g ZnSO₄•7H₂O 1.335 g  CoCl₂•6H₂O 0.13 gNiSO₄•H₂O 0.26 g CuSO₄•H₂O 0.01 g Na₂MoO₄•2H₂O 0.025 g  Deionized waterResidual

Example 2

Freeze Storage of the NBL-8 Strain

Colonies of the NBL-8 strain formed on an agar culture medium plate forThraustochytrid were collected with a sterilized inoculating loop andinoculated into 500 ml of a sterilized ribbed shaking flask containing100 ml of a liquid culture medium for Thraustochytrid. This flask wasshaken at 28° C. at 120 rpm for 2 to 5 days. TXY-25R-2F available fromTakasaki Kagaku Kikai Co., Ltd., (now, Preci Co., Ltd.) was used forculturing.

Subsequently, 20 ml of the obtained cultured cell suspension obtained byculturing in a shaking flask for 2 to 5 days was collected in 50 ml of asterilized test tube and centrifuged to recover cells. The recoveredcells were washed twice with artificial seawater. Subsequently, therecovered cells, that is, the NBL-8 strain, were suspended in 20 ml ofartificial seawater including a final concentration of 10 wt. % glyceroland 5 wt. % trehalose again. This served as a frozen cultured cellliquid of the NBL-8 strain and was frozen and stored at −80° C. untiluse in each experiment.

Example 3

Acquisition of the Mutant

0.1 ml of the frozen cultured cell liquid of the NBL-8 strain wasinoculated into 500 ml of a sterilized ribbed shaking flask containing100 ml of a liquid culture medium for Thraustochytrid. This flask wasshaken at 28° C. at 120 rpm for 2 days. 20 ml of the obtained culturedcell suspension after culture was transferred into a 50 ml sterilizedtest tube. Subsequently, cells in the cultured cell suspension wererecovered by centrifugation. The recovered cells were twice washed withsterile artificial seawater.

Next, in order to prompt the release of zoospores, the recovered cellswere shaken in artificial seawater. Specifically, 20 ml of the recoveredcells and artificial seawater were placed in a sterilized test tube andthe obtained cell suspension was shaken at 200 rpm for 30 minutes. Next,as a mutation inducing reagent, N-methyl-N′-nitro-nitrosoguanidine (NTG)was appropriately added to the cell suspension, so as to give a deathratio of 99% or higher, and shaken at 200 rpm for 1 to 30 minutes toinduce mutation. Cells subjected to the mutation inducing process wereappropriately diluted with artificial seawater, applied on an agarculture medium plate for Thraustochytrid, and cultured at 28° C. for 2to 5 days.

Colonies formed on the agar culture medium plate were selected with asterilized inoculating loop, after which each of the colonies wasstreaked onto a new agar culture medium plate for Thraustochytrid againand cultured. This operation was repeated twice to isolate the mutant.The cultured cell suspension was obtained from the isolated colonies asin Example 2. From 1 ml to 10 ml of this cultured cell suspension wascollected, centrifuged and frozen at −80° C. as in Example 2, thensupplied to a freeze drying machine (VA-140S available from TAITECCorporation) to obtain the freeze dried microbial body of a mutant.

The total lipid, that is, the microbial oil, was obtained from theobtained freeze dried microbial body and converted to a fatty acidmethyl ester.

Specifically, in accordance with the method (J. Biological andChemistry. 226:497-509(1957)) by Folch et. al., the total lipid wasextracted from the freeze dried microbial biomass using chloroform:methanol (2:1, v/v). The obtained total lipid was subjected to methylesterification to obtain a fatty acid methyl ester (FAME). FAME analysiswas carried out on the obtained FAME using gas chromatography. Theconditions of gas chromatograph were set as follows.

Column: DB-WAX of 0.530 mm×30 m, film thickness of 1.00 μm (AgilentTechnologies)

Carrier gas conditions: helium of 1.0 ml/minute, separation ratio of100:1

Column temperature conditions: at 140° C. for 5 minutes; the temperatureraised by 4° C./minute to 240° C.; and at 240° C. for 10 minutes

Detection: FID

Detector temperature: 260° C.

Inlet temperature: 250° C.

Injected quantity: 1 μL

As a result of the FAME analysis, 6 of 200 strains of the mutant withthe increased DHA concentration in the fatty acid were obtained. Amongthe parent strains having an indefinite circular colony shape and aundulate edge shape, those colonies having a more circular colony shape,an edge closer to the entire edge, and a smaller size were collected,with all of these colonies exhibiting common properties. Two among thesewere the NiD2 strain and the NiD3 strain. In accordance with the methoddescribed in Example 2, the obtained DHA concentration increasingstrains were cultured, frozen, and stored.

Example 4

Production of the DHA Microbial Oil

0.1 ml of the NBL-8 or NiD2 frozen cell liquid was inoculated into 500ml of a sterilized ribbed shaking flask containing 100 ml of a liquidculture medium for Thraustochytrid. This flask was shaken at 28° C. at120 rpm for 2 to 3 days. The obtained cell suspension served as theculture liquid to be inoculated in a jar fermenter.

0.5 L of the culture medium including 60 g glucose, 10 g yeast extract,15 g NaCl, 0.35 g KCl, 5.4 g MgCl₂.6H₂O, 2.7 g MgSO₄.7H₂O, 0.5 gCaCl₂.2H₂O, 1.0 mL of a vitamin solution, and 2.0 mL of a metal saltsolution was placed in a fermentation tank having a capacity of 1 L, and1% amount (v/v) of the culture liquid was inoculated therein.

Under the conditions of a culture temperature of 20° C., 26° C. or 32°C., a stirring speed of 600 rpm, an air flow rate of 1 vvm, an internalgauge pressure of 0 MPa, and a pH of 7.0±0.5, culturing was carried outfor 1 to 3 days. Subsequently, for 6 days, a feed liquid indicated inTable 6 was repeatedly added so as to give a glucose concentration of 6wt. % or less in the culture medium and continue culturing. In a casewhere a large amount of foaming occurred during culturing, an antifoamwas appropriately added. As the antifoam, a soybean oil, KM-72F, and thelike can be used.

TABLE 6 Feed liquid (1 liter) Glucose 500 g NaCl  15 g Ammonium Sulfate 9.8 g Monosodium Glutamate 27.8 g  Deionized water Residual

Subsequently, 2 ml of the cultured cell suspension was collected in a 2ml weighed centrifuge tube and centrifuged using a trace high speedcooling centrifuge to recover a microbial body. This washing process wascarried out several times and the microbial body was heated and dried at105° C. Subsequently, it was weighed to calculate the dry cell weight(DCW). DCW was measured three times to obtain the average value thereof.MX-300 available from Tomy Seiko Co., Ltd. was used as the trace highspeed cooling centrifuge.

Moreover, 10 ml of the cultured cell suspension was collected in a 15 mlcentrifuge tube, the freeze dried microbial body was obtained as inExample 2, the total lipid was obtained from the freeze dried microbialbody as in Example 3, the total lipid was subjected to methylesterification to obtain a fatty acid methyl ester, FAME analysis wascarried out by gas chromatography, and further, the fatty acid amountand DHA accumulative amount per dry microbial body were calculated.

0.1 ml of the frozen cell liquid of the NBL-8 or NiD2 was inoculatedinto a 500 ml sterilized ribbed shaking flask containing 100 ml of aliquid culture medium for Thraustochytrid. This flask was shaken at 22°C. and 20° C. at 120 rpm for 3 to 4 days.

Subsequently, it was weighed to calculate the dry cell weight (DCW). DCWwas measured three times to obtain the average value thereof.

The fatty acid composition (wt. %) of the fatty acid obtained from theNBL-8 or NiD2 strain, the amount of dried biomass per 1 liter of theculture liquid, the total fatty acid amount per 1 liter of the cultureliquid, the DHA amount per 1 liter of the culture liquid, and the DHAproduction efficiency are indicated in Table 7.

Hereinafter, also in the table, the amount of dried biomass per 1 literof the culture liquid is denoted by “DCW (g/L)”, the total fatty acidamount per 1 liter of the culture liquid is denoted by “TFA (g/L)”, theDHA amount per 1 liter of the culture liquid is denoted by “DHA (g/L)”,and the DHA production efficiency is denoted by “DHA (vsDCW).” Moreover,“others” in the table indicates the concentration of other fatty acids.

Regarding the concentration of various fatty acids in the fatty acidsproduced by the NiD2 strain cultured at 20° C., DHA was 50 wt. % ormore, while C16:0 was less than 30 wt. %. In contrast, regarding theconcentration of various fatty acids in the total fatty acid produced bythe NBL-8 strain cultured at 20° C., DHA was less than 40 wt. %, whileC16:0 was 30 wt. % or more.

Similarly, the DHA concentration in the fatty acids produced by the NiD2strain cultured at 26° C. or 32° C. was higher than the DHAconcentration in the fatty acids produced by the NBL-8 strain culturedat the same temperature.

The DHA production efficiency of all of the NiD2 strains was 20 wt. % ormore at the culture temperature, with each higher than the culturedNBL-8 strain at the same culture temperature.

Moreover, under all conditions, in the microbial oil of the NiD2 strain,the concentration of C12:0 was 0.1 wt. % or less.

The acid value (AV) in the microbial oil obtained from the NBL-8 strainand NiD2 strain, along with each concentration of the phospholipids,glycolipids, and unsaponifiable matter were determined in accordancewith the method described in the present specification, wherein the acidvalue (AV) was higher than 0.5 mg KOH/g, the phospholipid concentrationwas 3 wt. % or more, the glycolipid concentration was 3 wt. % or more,and the concentration of unsaponifiable matter was more than 4.5 wt. %.

Moreover, in the microbial oil of other microbial strains obtained as inthe microbial oil of the NiD2 strain, the triglyceride concentration was90 wt. % or more, the DHA concentration in triglyceride was 50 wt. % ormore, the phospholipid concentration was 5 wt. % or more, and theglycolipid concentration was 3 wt. % or more.

TABLE 7 Culture strain NBL-8 NiD2 Culture temperature (° C.) 20 26 32 2026 32 C14:0 3.4 4.3 3.9 3.6 3.0 2.2 C16:0 37.2 43.4 44.4 25.7 29.9 29.5C18:0 0.9 1.1 1.4 0.7 0.7 0.8 C22:5n-6 8.8 8.8 9.8 10.0 9.4 10.2C22:6n-3 45.4 38.3 35.2 51.2 47.1 44.4 others 4.3 4.0 5.3 8.9 10.0 12.9DCW(g/L) 88.8 84.2 84.2 70.6 65.9 51.7 TFA(g/L) 47.8 46.9 46.7 35.2 34.225.8 DHA(g/L) 21.7 18.0 16.5 18.0 16.1 11.4 DHA(vs DCW) 24.44% 21.38%19.60% 25.50% 24.43% 22.05%

Example 5

Production of High Content DHA by Low Temperature Culture

0.1 ml of the frozen cell liquid of the NiD2 strain was inoculated intoa 500 ml sterilized ribbed shaking flask containing 100 ml of a liquidculture medium for Thraustochytrid. This flask was shaken at 10° C. at120 rpm for 1 month. Subsequently, DCW measurement and FAME analysiswere carried out by the same method as in Example 4, and further, thefatty acid amount and DHA accumulative amount per dry microbial bodywere calculated.

The composition (wt. %) of the fatty acids produced by the NiD2 straincultured at 10° C., as well as the DHA concentration (wt. %) thereof,are indicated in Table 8.

The DHA concentration of the fatty acids produced at 10° C. was 70.5 wt.%. Moreover, the concentration of C12:0 was 0.1 wt. % or less.

TABLE 8 C14:0 1.0 C16:0 16.1 C18:0 0.6 C22:5n-6 8.9 C22:6n-3 70.5 others2.9

Example 6

Properties of the Microbial Oil

Microbial oils were each extracted as follows from part of the NBL-8 orNiD2 strain obtained in Example 4, as well as the NiD3 strain culturedunder the same conditions as in Example 2.

A cultured microbial body was heated and pasteurized, collected,centrifuged, washed, and heated and dried to obtain dry cells. 5-foldthe amount (w/v) of hexane was added to the dry cells to crush cellsusing a homogenizer. A suspension including this cell crushed matter wascentrifuged to recover a hexane layer including a microbial oil. Thisoperation was carried out three times. Subsequently, hexane wasdistilled off with an evaporator to obtain a microbial oil.

The obtained microbial oil was transferred into a sample bottle, the airin the upper part of the sample bottle was substituted with nitrogen,and the bottle was sealed. This microbial oil was stored at −80° C.until it was used for analysis.

With the extracted microbial oil serving as the target, the DSC pourpoint was measured as follows in accordance with the method described inthe document (Govindapillai et. al., (2009), Lubirication Science.21:13-26).

10 mg of a measurement sample was weighed in a sample pan using adifferential scanning calorimetry apparatus DSC 3500 Sirius (availablefrom NETZSCH), heated at 50° C. for 5 minutes, and cooled at a coolingspeed of 3° C./minute from 50° C. to −60° C.; subsequently, thetemperature was raised at a temperature raising rate of 10° C./minutefrom −60° C. to 50° C. for measurement to obtain the DSC curve. Thetemperature at the maximum value of the endothermic peak in the obtainedDSC curve was the DSC pour point (° C.).

The pour point was measured using the method described in TestingMethods for Pour Point and Cloud Point of Crude Oil and PetroleumProducts by the Japanese Industrial Standard (JIS K 2269-1987).

1000 mg of the microbial oil as the precipitate amount was collected ina weighed micro test tube having a capacity of 2 ml available fromEppendorf Japan and left to stand at 25° C. for 1 hour, after which theprecipitate was recovered using a trace high speed cooling centrifugeand the precipitate weight was measured using a precision balance,calculated as the wt. % in 1000 mg of the microbial oil. MX-300,available from Tomy Seiko Co., Ltd. was used as the trace high speedcooling centrifuge.

The fatty acid composition (wt. %), DSC pour point, pour point, andprecipitate amount of the microbial oil used in the experiment areindicated in Table 9. As indicated in Table 9, irrespective of theculture temperature, the microbial oil obtained from the NiD2 and NiD3strains had a low DSC pour point of 0° C. or lower, further had a lowpour point of 0° C. or lower, and had a smaller precipitate amount thanthe NBL-8 strain. Moreover, it was found that the NiD2 strain and NiD3strain have significantly lower DSC pour points than the NBL-8 strainserving as the parent strain and exhibit better handleability at lowtemperatures than the NBL-8 strain. Even in a case where the temperatureof the microbial oil thus obtained from the NiD2 strain and NiD3 strainis reduced from room temperature, fluidity tends not to change,resulting in excellent clearness as well as excellent handleability atlow temperatures.

It was found that because external measurement environment factors tendto have no influence during measurement using the DSC pour point, themeasured values are advantageously stable, and further, the use of theDSC pour point allows the properties of microbial oils based on thefatty acid composition to be grasped as the overall microbial oil moreaccurately than the pour point according to JIS K 2269-1987.

TABLE 9 NBL-8 NiD2 NiD3 Culture temperature 20 26 20 26 32 28 C14:0 3.44.1 3.8 3.0 2.2 3.0 C16:0 35.0 39.3 26.4 29.1 28.6 31.1 C18:0 0.9 1.10.7 1.0 1.2 1.2 C22:5n-6 9.1 9.5 10.4 10.1 10.9 12.0 C22:6n-3 47.5 41.953.1 50.3 47.9 48.0 others 4.1 4.3 5.5 6.4 9.1 4.6 DSC flow point (° C.)16.6 19.1 −9.1 −12.1 −10.4 −5.8 Flow point (° C.) 2.5 5.0 −10.0 −7.5−7.5 −5.0 Precipitate amount (%) 14 54 1 3 7 5

Next, when the properties of each of the microbial oils obtained byculturing the NiD2 strain or NBL-8 strain at 20° C. were subjected tosensory evaluation based on smoothness, the microbial oil derived fromthe NiD2 strain was smooth, giving it a preferable feel as a foodproduct. In contrast, in the microbial oil derived from the NBL-8strain, it felt rough, giving it unfavorable feel as a food product. Itwas found that the roughness of the microbial oil of the NBL-8 strain ispresumably due to crystals generated in the microbial oil, while in themicrobial oil derived from the NiD2 strain, the crystallization speed issuitably adjusted, and consequently, a favorable feel as a food productis obtained.

The microbial oil derived from the obtained NiD2 strain and NiD3 straincan be subjected to a refining process including the degumming step,deacidification step, decoloring step, and deodorizing step, to obtain arefined oil.

Moreover, the microbial oil derived from the obtained NiD2 strain andNiD3 strain, or the refined oil thereof can be subjected to the alkylesterification process by standard methods using a lower alcohol, toobtain a composition containing DHA alkyl ester. The microbial oilderived from the obtained NiD2 strain and NiD3 strain, or the refinedoil thereof, can be subjected to an alkaline decomposition process, forexample, with 5% sodium hydroxide at room temperature for 2 to 3 hours,after which a composition containing a free DHA can be obtained from theobtained decomposition liquid using a method regularly employed for theextraction or refining of fatty acids.

Furthermore, the thus obtained DHA alkyl ester containing composition orfree DHA containing composition can undergo a concentration process suchas distillation, rectification, column chromatography, the lowtemperature crystallization method, the urea clathrate method, orliquid-liquid countercurrent distribution chromatography to obtain aconcentrated DHA containing composition having a higher DHA content.

Therefore, the present disclosure enables the provision of a DHAcontaining oil, microbial oil, and biomass, which contain high contentDHA, have excellent stability at low temperatures, and exhibit favorablehandleability. Such a DHA containing oil, microbial oil, and biomass canbe preferably applied for uses such as in medicaments and supplements.

Disclosure of JP 2015-234985 filed on Dec. 1, 2015 is incorporatedherein in its entirety by reference.

All documents, patent applications, and technical specifications statedin the present specification are incorporated by citation in the presentspecification to the same degree as stated to be incorporated byreference specifically and individually.

What is claimed is:
 1. A docosahexaenoic acid containing microbial oilcomprising palmitic acid (C16:0) and stearic acid (C18:0), wherein themicrobial oil comprises (i) docosahexaenoic acid at a concentration of40 wt. % or more of the total weight of fatty acid in the microbial oil,(ii) palmitic acid (C16:0) at a concentration of 40 wt. % or less of thetotal weight of fatty acid in the microbial oil, and (iii) stearic acid(C18:0) at a concentration of 18.0 wt. % of less of the total weight offatty acid in the microbial oil, and wherein the microbial oil has anendothermic peak temperature, as determined by differential scanningcalorimetry (DSC), of 15° C. or lower.
 2. The docosahexaenoic acidcontaining microbial oil according to claim 1, wherein the endothermicpeak temperature, as determined by differential scanning calorimetry(DSC), is 10° C. or lower.
 3. The docosahexaenoic acid containingmicrobial oil according to claim 1, wherein the concentration of thedocosahexaenoic acid is from 40 wt. % to 98 wt. % of the total weight offatty acid in the microbial oil.
 4. The docosahexaenoic acid containingmicrobial oil according to claim 1, wherein the endothermic peaktemperature, as determined by differential scanning calorimetry (DSC),is from −50° C. to 10° C.
 5. The docosahexaenoic acid containingmicrobial oil according to claim 3, wherein the concentration of thedocosahexaenoic acid is from 70 wt. % 70 wt. % to 98 wt. % of the totalweight of fatty acid in the microbial oil.
 6. The docosahexaenoic acidcontaining microbial oil according to claim 1, wherein the microbial oilis obtained from at least one microorganism selected from the groupconsisting of: Opisthokonta, Archaeplastida, Excavata, SAR, amicroorganism belonging to Haptophyta and Cryptophyta not classified asthe aforementioned, and a bacterium.
 7. The docosahexaenoic acidcontaining microbial oil according to claim 1, wherein the microbial oilis obtained from a microorganism belonging to Stramenopiles.
 8. Thedocosahexaenoic acid containing microbial oil according to claim 1,wherein the microbial oil is obtained from a microorganism belonging tothe class Labyrinthulea.
 9. The docosahexaenoic acid containingmicrobial oil according to claim 1, wherein the microbial oil isobtained from a microorganism belonging to a Thraustochytridmicroorganism.
 10. The docosahexaenoic acid containing microbial oilaccording to claim 1, wherein the microbial oil is obtained from amicroorganism of the genus Aurantiochytrium.
 11. The docosahexaenoicacid containing microbial oil according to claim 10, wherein themicrobial oil is obtained from a docosahexaenoic acid producing mutantof Aurantiochytrium limacinum.
 12. The docosahexaenoic acid containingmicrobial oil according to claim 11, wherein the microbial oil isobtained from an Aurantiochytrium limacinum strain selected from thegroup of strains consisting of: a NiD2 strain (accession number FERMBP-22296), a NiD3 strain (accession number FERM BP-22297) a microbialstrain having substantially the same microbiological properties as theNiD2 or NiD3 strain.
 13. The docosahexaenoic acid containing microbialoil according to claim 1, wherein the microbial oil is a crude oil. 14.The docosahexaenoic acid containing microbial oil according to claim 1,wherein the microbial oil is a refined oil.
 15. A biomass ofmicroorganisms, the biomass comprising the docosahexaenoic acidcontaining microbial oil according to claim
 1. 16. The biomass accordingto claim 15, wherein the accumulative amount of the docosahexaenoic acidis 18 wt. % or more of the dried biomass weight per 1 liter of a cultureproduct.
 17. The biomass according to claim 15, wherein the biomass is adried biomass.
 18. A microorganism of the genus Aurantiochytrium capableof producing the docosahexaenoic acid containing microbial oil accordingto claim
 1. 19. The microorganism of the genus Aurantiochytriumaccording to claim 18, wherein the microorganism is an Aurantiochytriumlimacinum strain selected from the group of strains consisting of a NiD2strain (accession number FERM BP-22296) and a NiD3 strain (accessionnumber FERM BP-22297).
 20. The microorganism of the genusAurantiochytrium according to claim 18, having substantially the samemicrobiological properties as an Aurantiochytrium limacinum NiD2 strain(accession number FERM BP-22296) or an Aurantiochytrium limacinum NiD3strain (accession number FERM BP-22297).
 21. A manufacturing method of abiomass comprising a docosahexaenoic acid containing microbial oil, themethod comprising: culturing the microorganism of the genusAurantiochytrium of claim 18; and recovering the biomass from thecultured microorganism.
 22. The method of biomass according to claim 21,the method further comprising drying the biomass obtained after culture.23. A manufacturing method of docosahexaenoic acid containing microbialoil, the method comprising: culturing the microorganism of the genusAurantiochytrium according to claim 18 to obtain a biomass; recoveringthe biomass from the cultured microorganism; and extracting oil from therecovered biomass to obtain an extracted oil, wherein a docosahexaenoicacid containing microbial oil comprising docosahexaenoic acid at aconcentration of 40 wt. % or more of the total weight of fatty acid inthe oil is obtained from the extracted oil, and the docosahexaenoic acidcontaining microbial oil has an endothermic peak temperature asdetermined by differential scanning calorimetry (DSC), of 15° C. orlower.
 24. The method according to claim 23, wherein the obtaineddocosahexaenoic acid containing microbial oil is a crude oil.
 25. Themethod according to claim 23, the method further comprising refining theextracted oil.
 26. The method according to claim 21, wherein themicroorganisms are cultured at 10° C. to 40° C.
 27. The docosahexaenoicacid containing microbial oil according to claim 1, wherein theconcentration of the palmitic acid (C16:0) is from 5.0 wt. % to 40 wt. %of the total weight of fatty acid in the microbial oil.
 28. Thedocosahexaenoic acid containing microbial oil according to claim 1,wherein the concentration of the stearic acid (C18:0) is from 0.3 wt. %to 18.0 wt. % of the total weight of fatty acid in the microbial oil.